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Mechanical stimulation of plants can be caused by various abiotic and biotic environmental factors. Apart from the negative consequences, it can also cause positive changes, such as acclimatization of plants to stress conditions. Therefore, it is necessary to study the physiological and biochemical mechanisms underlying the response of plants to mechanical stimulation. Our aim was to evaluate the response of model plant Arabidopsis thaliana to a moderate force of 5 N (newton) for 20 s, which could be compared with the pressure caused by animal movement and weather conditions such as heavy rain. Mechanically stimulated leaves were sampled 1 h after exposure and after a recovery period of 20 h. To study a possible systemic response, unstimulated leaves of treated plants were collected 20 h after exposure alongside the stimulated leaves from the same plants. The effect of stimulation was assessed by measuring oxidative stress parameters, antioxidant enzymes activity, total phenolics, and photosynthetic performance. Stimulated leaves showed increased lipid peroxidation 1 h after treatment and increased superoxide dismutase activity and phenolic oxidation rate after a 20-h recovery period. Considering photosynthetic performance after the 20-h recovery period, the effective quantum yield of the photosystem II was lower in the stimulated leaves, whereas photochemical quenching was lower in the unstimulated leaves of the treated plants. Nonphotochemical quenching was lower in the stimulated leaves 1 h after treatment. Our study suggested that plants sensed moderate force, but it did not induce pronounced change in metabolism or photosynthetic performance. Principal component analysis distinguished three groups–leaves of untreated plants, leaves analysed 1 h after stimulation, while stimulated and unstimulated leaves of treated plants analysed 20 h after treatment formed together the third group. Observed grouping of stimulated and unstimulated leaves of treated plants could indicate signal transduction from the stimulated to distant leaves, that is, a systemic response to a local application of mechanical stimuli.

In Arabidopsis thaliana (L.) Heynh., a major part of MATH-BTB protein family (BPM proteins) participate in the ubiquitin‐proteasome pathway as substrate-specific adaptors of Cullin 3 dependent E3 ligase. Through targeting of specific proteins for degradation, BPMs are involved in various developmental processes, as well as in the plant’s ability to adapt to changing environmental conditions. The aim of this research was to investigate the oxidative stress response of Arabidopsis with modified BPMs expression to moderate heat stress. Seedlings of wild type, line overexpressing BPM1 gene (oeBPM1), and line with down-regulation of BPM1, 4, 5, and 6 genes (amiR bpm) were exposed to 37 °C for six hours. Seedlings were sampled immediately after stress and after a recovery period of 24 h. Control seedlings were grown at 24 °C and were collected at the same time points as treated ones. The level of lipid peroxidation, H2O2 and proline content, as well as the activity of antioxidant enzymes (G POD, APX, CAT, and SOD) were evaluated. Control amiR bpm and oeBPM1 seedlings generally had lower H2O2 and proline content, respectively, than wild type. After exposure to 37 °C, oeBPM1 and amiR-bpm lines showed reduced proline content, while no change in lipid peroxidation level and H2O2 content was observed. Further, seedlings with modified BPMs expression showed differences in the activity of G POD, APX, and SOD, while no change was observed in the activity of CAT. The results obtained suggest involvement of BPM proteins in the response of Arabidopsis to moderate heat stress.

Glavna uloga proteina BPM1 u uročnjaku (Arabidopsis thaliana L.) je u kompleksu kulin3- ovisnih E3 ligaza gdje domenom MATH prepoznaje specifične proteinske supstrate usmjeravajući ih u ubikvitinaciju i degradaciju na proteasomu 26S. Metodama ko-imunoprecipitacije i spektrometrije masa utvrđena je interakcija proteina BPM1 s DMS3 i RDM1 komponentama kompleksa DDR uključenog u RNA-posredovanu metilaciju DNA (RdDM). Kompleks DDR regrutira Polimerazu V te sudjeluje u pozicioniranju metilacijskog kompleksa na genom. Navedene interakcije nisu posredovane domenom MATH, ukazujući kako se kroz ovu interakciju proteini DMS3 i RDM1 ne degradiraju. U svrhu razjašnjena uloge proteina BPM1 u RdDM-u, odabrano je pet gena za analizu metilacije bisulfitnim mapiranjem i pirosekvenciranjem. Geni FWA i CML41 korišteni su kao kontrole budući da je njihova regulacija RdDM-om poznata od ranije. Kromatinskom imunoprecipitacijom identificirali smo zajedničke vezne regije proteina BPM1 i DMS3 na genomu linije s nefunkcionalnom proteasomalnom degradacijom, te izdvojili gene AGL14, RKP i FBW2 kao nove mete proteina BPM1 i RdDM-a. Metilacijski obrasci za svako metilacijsko mjesto i metilacijski kontekst, uspoređeni su između divljeg tipa te linije s overekspresijom proteina BPM1 i linije s nefunkcionalnim proteinom DMS3. Rezultati su pokazali značajno više razine metilacije povezane s RdDM-om u biljkama s povišenom ekspresijom proteina BPM1 te gubitak metilacije u RdDM nefukcionalnoj mutanti dms3.

U biljaka proces embriogeneze, može započeti iz oplođene jajne stanice (zigotna embriogeneza), ali i indukcijom somatskih stanica (somatska embriogeneza). U oba slučaja dolazi do značajnog epigenetičkog reprogramiranja, a jedan od ključnih mehanizama je metilacija DNA posredovana malim molekulama RNA (engl. RNA-directed DNA methylation, RdDM) koja osigurava metilaciju de novo. Uročnjakov protein BPM1 dio je velike porodice proteina MATH-BTB koji sudjeluju u brojnim razvojnim procesima biljaka i životinja kao komponente proteina ubikvitinskih ligaza ovisnih o kulinu. Dodatno, protein BPM1 stupa u interakciju s proteinima DMS3 i RDM1, važnim komponentama u procesu RdDM-a. Interakcije se ostvaruju neovisno o kulinu, upućujući na ulogu proteina BPM1 u regulaciji metilacije DNA de novo. Kromatinskom imunoprecipitacijom identificirane su zajedničke vezne regije proteina BPM1 i DMS3 na genomu uročnjaka te su odabrani geni na kojima su provedene analize ekspresije i metilacije u somatskim i zigotnim embrijima uročnjaka. Također, ispitana je učinkovitost indukcije somatskih embrija u transgeničnim linijama s prekomjernom ekspresijom proteina BPM1 ili DMS3, u liniji s utišanim genima BPM te liniji s nefunkcionalnim proteinom DMS3. Embriogeni potencijal linije s utišanim genima BPM bio je smanjen, dok u linijama s prekomjernom ekspresijom nije došlo do značajnih promjena u odnosu na divlji tip. Dobiveni rezultati upućuju na novu ulogu proteina MATH-BTB u regulaciji metilacije DNA tijekom embriogeneze.

The protein DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) is one of the key components of RNA-directed DNA methylation (RdDM). By mediating DNA methylation, RdDM has a role in controlling genome integrity and gene expression, particularly during plant acclimation to fluctuating environmental conditions. The aim of this study was to question the impact of elevated temperature on selected physiological and biochemical parameters in Arabidopsis thaliana with modified DMS3 expression. Seedlings with overexpressed DMS3 gene (oeDMS3 line) or non-functional DMS3 protein (dms3-1 line) together with the wild type (wt), were cultured at temperature of 24 °C and subsequently exposed to 37 °C for six hours. Plant material was collected at two time points - immediately after the treatment and after a 24-hour recovery period at 24 °C. In dms3-1 line, exposure to 37 °C resulted in a decrease of photosynthetic performance at both time points. Increased proline and MDA content were observed only in dms3-1 after recovery. In oeDMS3 line, guaiacol peroxidase (G-POD), ascorbate peroxidase (APX), and catalase (CAT) activity increased immediately after the treatment, whereas superoxide dismutase (SOD) increased after recovery. In dms3-1 line, only CAT activity increased immediately after the treatment, whereas G POD decreased at both time points. It can be concluded that dms3-1 was more heat-sensitive than oeDMS3 and wt, suggesting that the DMS3 protein is required for heat tolerance in Arabidopsis.

With ongoing climate change, rising temperatures are becoming a severe threat to crop yield and food security worldwide. Owing to their sessile lifestyle, plants had evolved many ways to circumvent their apparent handicap. Complex molecular machineries deal with sensing, transmittance and response to environmental stimuli. To evaluate the impact of heat stress (HS), the globally cultivated tomato (Solanum lycopersicum) was chosen as a model plant. Tomato germination potential was investigated at a temperature range from 24 to 36 °C. Seedling treatments consisted of prolonged exposure to 37 °C and a short-term exposure to 45 °C. Root growth and seedling morphology were determined. For HS evaluation, proline and malondialdehyde (MDA) content were measured, HSP90 immunoassayed and HS-related gene expression analyzed by quantitative PCR. The maximal permissive germination temperature for tomato seeds was 34 °C. Both HS regimes caused increased levels of HSP90 and HS-related gene expression in seedlings. Prolonged exposure to 37 °C did not, in contrast to the 45 °C-treatment, significantly affect proline and MDA levels, but inhibited root and shoot growth. The heat wave-like treatment at 45 °C completely blocked primary root growth and strongly inhibited seedling development. Inclusive, both types of exposures negatively affected growth, biochemical and molecular pathways, indicating high sensitivity of tomato seedlings to applied HS treatments.

Fitoplazme (rod 'Candidatus Phytoplasma') su fitopatogene bakterije bez stanične stijenke, obligatni unutarstanični paraziti floema biljaka i kukaca. Uzročnici su bolesti i gubitaka prinosa inih usjeva u svijetu. Prijenos fitoplazmi kukcima-vektorima uglavnom ovisi o površinskim fitoplazmatskim proteinima. Za antigenski membranski protein (AMP) 'Ca. P. asteris' pokazano je da stupa u interakciju s proteinom aktinom, mikrofilamentom kukaca. Stamp je antigenski membranski protein pronađen samo u ‘Ca. P. solani’. Njegova aminokiselinska sekvenca podudara se s proteinom AMP ‘Ca. P. asteris’ 26 – 40%, a sa sekvencom AMP ‘Ca. P. japonicum' 40%. S obzirom na sličnosti i moguće uloge ovih proteina, cilj istraživanja bio je ispitati protein – protein interakciju fitoplazmatskog proteina Stamp porijeklom iz vinove loze (V. vinifera) i aktina (HoACT) porijeklom iz kukca-vektora Hyalesthes obsoletus. Dizajnirani su ekspresijski plazmidi s ugrađenim cjelovitim i mutiranim genima stamp ST6 i ST9, te plazmidi s genom HoACT kako bi se istražila njihova interakcija. Istraživanje je obuhvaćalo korištenje bimolekularne fluorescencijske komplementacije (BIFC) in planta i metodu kopurifikacije proteina pull-down. Rezultati kopurifikacije proteina su dokazali interakciju hidrofilne regije proteina Stamp i HoACT. Metodom BIFC u listovima duhana Nicotiana benthamiana interakcija nije potvrđena te je metodu potrebno prilagoditi kako bi se razjasnili uvjeti interakcije proteina in planta.

In the Arabidopsis thaliana (L.) Heynh. genome, six BPM genes encode proteins containing BTB/POZ (Bric-a-Brac, Tramtrack, Broad Complex) and MATH (Meprin and TRAF Homology) domains. Recent studies have shown that BPM proteins play an important role in heat stress through the negative regulation of DREB2A, a transcription factor controlling the expression of many genes in response to drought and heat stress. Moreover, BPM1 protein was shown to be stabilized and accumulated under moderate heat stress. Physiological response of Arabidopsis seedlings with modified BPMs expression to moderate heat stress was investigated in wild type (wt), the line overexpressing BPM1 gene (oeBPM1) and the line with downregulation of BPM1, 4, 5 and 6 genes (amiR-bpm). Seedlings were exposed to 37 °C for six hours. Plant material was collected at two time points – immediately after exposure to elevated temperature and after a recovery period of 24 h at the cultivating temperature (24 °C). The status of photosynthetic apparatus was analyzed by chlorophyll a fluorescence transient (OJIP test). The content of pigments (carotenoids, chlorophyll a and b), proline, hydrogen peroxide (H2O2) and level of lipid peroxidation, as well as the activity of the antioxidant enzymes catalase, superoxide dismutase, guaiacol and ascorbate peroxidases were measured spectrophotometrically. Immediately after exposure to 37 °C, seedlings of all three lines showed reduced proline content compared to the non-stressed control groups. The photosynthetic performance index and pigment content were decreased in the wt and oeBPM1 immediately after stress. Seedlings with altered BPMs expression showed different dynamics of H2O2, lipid peroxidation and antioxidant enzymes. Different physiological response of wt, oeBPM1 and amiR-bpm to moderate heat stress could be related to altered expression of BPMs in examined lines.

Temperatures vary geographically and are predicted to rise with global warming. As sessile organisms, plants are unmercifully exposed to such environmental conditions causing crop loss worldwide. For dormant seeds, temperature and light are two main cues required for germination (Donhoe et al. 2003). Therefore, appropriate germination timing and response to temperature cues are necessary for plant development. A rich repertoire of flexible epigenetic machineries that control gene expression enables plants a quick response to external signals and to readily adapt to environmental conditions (Centomani et al. 2015). Understanding the epigenetic basis of sensitivity to temperature is therefore required for understanding interactions between plants and environment. Considering the importance of seeds for agriculture, we aimed to evaluate the impact of different heat stress regimes during the process of germination, partially mimicking global warming conditions. We determined the maximum permissive germination temperature and global DNA methylation levels of three plant species: model plant Arabidopsis thaliana and two globally important crop species Brassica rapa and Solanum lycopersicum.

Temperature is one of the major parameters influencing plant life alongside relative humidity and light. It varies geographically and changes with time. Here, the effect of heat stress on growth and physiology of model plant Arabidopsis thaliana (thale cress), and two globally important crop species, Solanum lycopersicum (tomato) and Brassica rapa (chinese cabbage) was investigated. Seedlings were exposed to a moderate elevated temperature of 37 °C, a temperature common for the summer season in the Mediterranean, and to a severe elevated temperature of 45 °C. This extreme temperature regime was chosen based on literature data and the highest recorded temperature in Croatia [1, 2]. Timeframes of seedling survival at such an extreme condition were evaluated. In heat treated seedlings, prolin content and levels of lipid peroxidation were measured. Both parameters show an increase in heat treated seedlings in comparison to controls. As a consequence of global warming, environmental temperatures have risen in recent years and continue to do so. Thus, these results, together with previous research, will provide a clearer picture of heat tolerance and response in plants.

Voda, temperatura i svjetlost neophodni su za prekid dormancije i klijanje sjemena. Zbog klimatskih promjena, vegetacijsko razdoblje sve je sušnije, prosječna je temperatura u porastu, a češći su i toplinski udari. Promjena klime koju očekujemo u nadolazećem razdoblju utjecat će na poljoprivredu i značajno ugroziti bioraznolikost. Biljke imaju brojne mehanizme koji im omogućuju da se prilagode uvjetima okoline, a osnova tih promjena je epigenetičke prirode. Stoga je za razumijevanje interakcija između biljaka i okoliša potrebno razumijevanje epigenetičke osnove osjetljivosti na temperaturu. U ovom je radu istraženo kako temperatura utječe na klijanje triju biljnih vrsta - modelne biljke uročnjaka (Arabidopsis thaliana) i dviju globalno važnih poljoprivrednih vrsta: kineskog kupusa (Brassica rapa) i rajčice (Solanum lycopersicum). Za svaku je vrstu istraženo koja je najviša temperatura koja omogućuje klijanje sjemenki, kao i kako povišenje temperature utječu na globalnu metilaciju DNA.

A member of a small family of Arabidopsis thaliana MATH-BTB proteins, BPM1 functions as a substrate-specific adaptor of a cullin3-based E3 ubiquitin ligase complex. Its N-terminal MATH domain binds target proteins and designates them for ubiquitination and subsequent proteasomal degradation. Recent studies reported that BPM1 mediates proteasomal degradation of transcription factors (ERF/AP2, R2R3 MYB and Homeobox) and phosphatases (PP2Cs). By using immunoprecipitation, TAP-tag purification, fluorescence-lifetime imaging microscopy, yeast-2-hybrid, pull down and microscale thermophoresis we confirmed BPM1 interaction with DMS3 and RDM1, key regulators of de novo DNA methylation in A. thaliana. Chromatin immunoprecipitation was used to determine regions of overlap between DMS3 and BPM1 chromatin binding sites. The DNA methylation status of identified regions was analysed by pyrosequencing after bisulphite conversion in A. thaliana plants overexpressing BPM1, in plants with downregulated BPMs as well as in DNA methylation mutants. Methylation patterns were assessed during plant development and after exposure to different abiotic stresses. Our results indicate that BPM1 does not participate in degradation of DMS3 and RDM1. Rather, it seems BPM1 recruits DNA methylation machinery to specific chromatin positions for de novo DNA methylation establishment.

Proteini MATH-BTB sadrže 2 domene, MATH (Meprin and TRAF Homology) i BTB (Bric-A-Brac, Tramtrack, Broad Complex) i opisani su kao adapteri u kompleksu E3 ligaza ovisnih o Cul3. Proteini MATH-BTB u sklopu ovog kompleksa djeluju kao sustav navođenja ciljanih proteina na ubikvitinaciju, a zatim i degradaciju na proteasomu 26S. U genomu uročnjaka Arabidopsis thaliana 6 gena kodira proteine MATH-BTB, nazvane AtBPM1-6. Dosad opisane mete proteina AtBPM transkripcijski su faktori iz porodice R2R3 MYB, ERF/AP2 i razreda I HD-Zip, čime su proteini AtBPM dovedeni u vezu s procesima poput regulacije cvjetanja (1), razvoja sjemena (2) i odgovora na abiotički stres (3). Transkripcijski faktor DREB2A regulira odgovor biljke na toplinski stres aktivacijom ekspresije velikog broja nizvodnih gena, među kojima je i gen HsfA3, a nedavno je pokazano da proteini AtBPM ulaze u interakciju s proteinom DREB2A te reguliraju njegovu razgradnju (4). S ciljem istraživanja fiziološke uloge proteina BPM1, proizvedene su transgene biljke A. thaliana koje prekomjerno eksprimiraju protein BPM1 fuzioniran sa zelenim fluorescentnim proteinom (GFP). Klijanci transgene linije uročnjaka izlagani su povišenim temperaturama, a prisutnost i distribucija proteina AtBPM1 detektirana je konfokalnom mikroskopijom. Iz tkiva tretiranih klijanaca izolirani su ukupni topivi proteini te je protein GFP-BPM1 analiziran imunodetekcijom pomoću anti-GFP antitijela. Dok je u kontrolnim uvjetima zabilježena degradacija proteina GFP-BPM1, izlaganje povišenim temperaturama potaknulo je značajnu stabilizaciju proteina GFP-BPM1. Kvantitativnom metodom PCR analizirana je ekspresija gena HsfA3 u transgenim klijancima izloženima povišenoj temperaturi (37 °C). U odnosu na divlji tip, ekspresija gena HsfA3 bila je značajno smanjena. Zajedno, ovi rezultati ukazuju na potencijalnu fiziološku ulogu proteina BPM1 u prilagodbi uročnjaka na povišene temperature.

Proteini BPM uročnjaka pripadaju porodici proteina MATH-BTB koji sudjeluju u brojnim razvojnim procesima biljaka i životinja, te su se pokazali ključnim regulatorima staničnog ciklusa. Genom uročnjaka sadrži šest gena koji kodiraju proteine BPM (BPM1-6). Uročnjakovi proteini BPM djeluju kao specifični adapteri za kulin3 koji je dio kompleksa ubikvitinskih ligaza E3. Proteini BPM vežu kulin3 svojom domenom BTB, dok domena MATH prepoznaje specifične supstrate poput transkripcijskih faktora te ih usmjerava na ubikvitinaciju i posljedičnu proteasomsku razgradnju [1]. Unutarstanična lokalizacija proteina BPM1 u jezgri, ali i jezgrici upućuje na funkciju ovog proteina neovisnu o kulinu3 [2]. Preliminarna istraživanja pokazala su da protein BPM1 ulazi u interakciju s proteinima DMS3 i RDM1, važnim komponentama u procesu metilacije DNA de novo usmjerene malim molekulama RNA (RdDM). S ciljem razjašnjavanja funkcije proteina BPM1 u procesu RdDM analizirana je unutarstanična kolokalizacija proteina BPM1 s DMS3 i RDM1 te značaj pojedinih proteinskih domena u ostvarivanju specifične unutarstanične lokalizacije. Sustavom dvaju kvašćevih hibrida istražene su interakcije šest proteina BPM i varijanti proteina BPM1 bez pojedinačne proteinske domene s proteinima DMS3 i RDM1. Rezultati ukazuju da proteini BPM ostvaruju interakcije s DMS3 i RDM1, te da je domena BTB važna za interakciju s RDM1. Protein BPM1 primarno lokalizira u jezgri u kojoj je potvrđena njegova kolokalizacija s proteinima DMS3 i RDM1. Delecija pojedinih domena proteina ne narušava kolokalizaciju proteina BPM1 u jezgri, ali se odražava na lokalizaciju u jezgrici. Interakcije proteina BPM1 s proteinima DMS3 i RDM1 unutar jezgre upućuju na potencijalnu ulogu proteina BPM1 u regulaciji metilacije DNA de novo regulirane mehanizmom RdDM. Nadalje, značaj domene BTB u pokazanim interakcijama ukazuje da mehanizam kojim protein BPM1 sudjeluje u putu RdDM nije temeljen na usmjeravanju regulacijskih proteina RdDM (DMS3 i RDM1) u razgradnju na proteasomima, a rasvjetljavanje mehanizma opisane interakcije u putu RdDM-a predmet je daljnjih istraživanja.

Temperatura je važan čimbenik za rast i razvoj biljaka. Ona varira s geografskom širinom, ali se također mijenja kao posljedica globalnog zatopljenja. Vlaga, svjetlost i temperatura tri su čimbenika presudna za germinaciju, a pored hormonskog statusa, sinteze i degradacije proteina tijekom germinacije, presudnu ulogu u adaptaciji biljaka na okolišne uvjete imaju epigenetičke promjene. U svrhu proučavanja učinka povišene temperature na metilaciju DNA istražena je sposobnost germinacije modelne biljke Arabidopsis thaliana (uročnjak), te dviju poljoprivredno važnih vrsta Nicotiana tabacum (duhan) i Brassica rapa (poljska vrzina) u uvjetima povišene temperature. Za germinacijske su testove sjemenke navedenih vrsta izložene setu temperaturnih tretmana od 28 do 48 °C te je određena maksimalna permisivna temperatura klijavosti. U uzorcima izloženim povišenim temperaturama odredit će se stupanj metilacije DNA.

A native Croatian grapevine 'Grk bijeli', sharing a parent-offspring relationships with 'Tribidrag' (aka 'Zinfandel'), is grown exclusively on the Adriatic island of Korčula. It is one of the grape cultivars with female-only functional flowers, causing reduced fertilisation and leads to problems in grape production and wine quality. A typical cluster of 'Grk bijeli' at maturity contains fully developed seeded berries, in addition to a highly variable proportion of undersized seedless berries. The aim of this study was to identify the cause of the reduced reproduction potential of 'Grk bijeli' in order to establish a better growing environment for improved yield and grape quality. 'Grk bijeli' female gametophyte develops normaly and at maturity contains both egg and central cell, together with two synergid cells and three antipodal cells. On the other hand, 'Grk bijeli' pollen grains show developmental deviations. Specifically, most of the pollen grains undergo the first pollen mitosis and contain one vegetative cell and one generative cell, while 20 % of ungerminating grains also accomplish the second pollen mitosis, giving rise to two sperm cells and one vegetative cell. Morover, 'Grk bijeli' pollen has acolporate morphology, which prevents germination and contributes to reduced reproduction. Furthermore, fertilisation after pollination with other varieties results in various degrees of ovule abortion depending on the pollinator, revealing Croatian cultivars 'Plavac mali crni' and 'Pošip bijeli' as favourable varieties. Although this study describes a highly valuable cultivar of local importance, it also contributes to fundamental knowledge of grapevine reproductive biology and offers a strategy for improvement of wine production and oenological performance of semi-fertile varieties in general., VITIS - Journal of Grapevine Research, Vol 58 No 2 (2019): Vitis

Methylation of a DNA molecule is one of several epigenetic mechanisms for controlling gene expression in cells, and acts as a signal to "turn" on and off the genes. In addition to regulating developmental processes in plants, the diversity of epigenetic mechanisms plays an important role in the phenotypic and physiological adaptability as well as the ability of plants to survive and reproduce in unpredictably changing environments. One of the basic epigenetic specificities of plants is the mechanism of RNA-directed DNA methylation (RdDM) de novo DNA methylation. RdDM requires a specialized transcription mechanism consisting of two plant-specific RNA polymerases - Pol IV and Pol V - and a large number of accessory proteins, but the mechanism is not fully elucidated. One hypothesis that will be discussed is the potential of RdDM in balancing the active and inactive status of target genes.

Arabidopsis thaliana BPM1 protein belongs to six-membered MATH-BTB protein family. The best- known function of MATH-BTB proteins is their role as substrate-specific adaptors of cullin3- based E3 ligases in the ubiquitin-proteasome pathway. BTB domain enables assembly with the cullin while less conserved MATH domain serves as an adaptor and binds substrates destined for degradation via 26S proteasome. Targets of BPM1 include several transcription factors such as WRI1, HB6, DREB2A and MYB6. Through mediation of degradation of these transcription factors BPM1 is involved in the regulation of various plant processes including fatty acid metabolism, abscisic acid signalling regulation, flowering etc. Earlier results showed that BPM1 predominantly localizes in nucleus indicating cullin independent function of BPM1. Co-immunoprecipitation and mass spectrometry revealed interaction of BPM1 with DMS3 and RDM1, key components of RNA-directed DNA methylation (RdDM). However, exact role of BPM1 interaction with DMS3 and RDM1 and possible implications for RdDM pathway remain unknown. To furthermore elucidate putative role of BPM1 protein in RdDM yeast two hybrid and pull down assays were used to confirm direct interactions of BPM1 with DMS3 and RDM1. Additionally, role of MATH and BTB domain in the interaction with DMS3 and RDM1 was tested using truncated version of BPM1 protein with a single domain deletion. The results showed that BTB domain had higher affinity for interaction with RDM1, while both MATH and BTB domains appear to be equally important for interaction with DMS3. In addition, co-localization assay showed overlap of BPM1 and RdDM components while overexpression of BPM1 revealed change in DNA methylation pattern. These results indicate a novel role of BPM1 protein in RNA-directed DNA methylation pathway. Our next aim is to use ChIP in order to identify DNA sites where transcription or RdDM methylation could be influenced by BPM1 protein.

Arapidopsis BPM proteins belong to MATH-BTB protein family. MATH-BTB proteins partake in many developmental processes in plants and animals and are known as key regulators of the cell cycle. Arabidopsis genome contains six genes encoding for BPM proteins (BPM1-6). BPM proteins bind members of several transcription factor families targeting them for ubiquitination by Cullin3 E3 ligase. Our preliminary results showed BPM1 interaction with DMS3 and RDM1, important components of RNA-directed DNA methylation (RdDM). Still, the role of BPM proteins in DNA methylation remains unknown. To elucidate an involvement of other Arabidopsis MATH-BTB proteins (BPM2-6) in RdDM, interactions with DMS3 and RDM1 were examined in yeast two hybrid system. Additionally, truncated versions of the BPM1 missing an individual domain were used to test domain specificity for interaction with DMS3 and RDM1. All BPM proteins show interactions with both investigated components of RdDM. BTB domain of BPM1 protein proved to be the most important for interaction with RDM1. Results obtained here point to a role of BPM proteins in regulation of DNA methylation that will be further investigated.

MATH-BTB proteins are comprised of two domains, MATH (Meprin and TRAF Homology) and BTB (Bric- A-Brac, Tramtrack, Broad Complex). While the MATH domain recognizes specific substrates, BTB domain binds Cullin 3 (CUL3) of the E3 ubiquitin ligase complex which ubiquitinates and thereby designates target proteins for proteasomal degradation. The MATH-BTB gene family is represented by six members in the Arabidopsis genome (AtBPM1-6). AtBTB proteins interact with members of at least three families of transcription factors (R2R3 MYB family, ethylene response factor/Apetala2 and class I homeobox-leucine zipper transcription factors) which play important roles during plant flowering, seed development and abiotic stress response. Here, transgenic A. thaliana plants with overexpression of GFP-tagged BPM1 were successfully regenerated and analyzed, exhibiting early flowering phenotype and resistance to ABA. Despite high overexpression rate, recombinant BPM1 protein was unstable under control conditions. The stability of BPM1 protein was analyzed after treatment of transgenic seedlings in conditions of salt and osmotic stress (150 mM NaCl or 300 mM mannitol), darkness, elevated temperatures, and upon heat stress. Seedlings were collected for whole soluble protein extraction and anti-GFP monoclonal antibody was used to blot transgenic GFP-tagged proteins. Darkness and salt stress induced degradation of GFP-BPM1, as opposed to heat treatment which caused significant accumulation of GFP-BPM1. Improved stabilization of transgenic GFP-BPM1 in conditions of elevated temperature could indicate the physiological role of AtBPM1 in response to heat stress.

The steady increase in temperature causes worldwide losses in agriculture, therefore heat stress is a problem in many areas in the world. Tomatoes are a major horticultural crop worldwide. Their yields are maximized in climates with moderate temperatures, whereas high temperature exposure limits tomato flower development, fruit set, and thus productivity. Experimental evidence indicates that reduced fruit set at high temperatures results from inhibited pollen development, anther release, and pollen viability reduction correlating with altered metabolism of the plant hormone auxin. We have therefore set out to analyze the auxin metabolome and transcriptome in tomato flower and fruit organs to identify candidates for alteration of auxin homeostasis. Among the strongly differentially regulated genes, four GH3 genes encoding auxin conjugate synthetases were specifically upregulated in male flower organs and gametophyte during flower development of tomato. We cloned these for heterologous expression in Escherichia coli to determine their in vitro activities. One highly regulated gene encoding an amino acid conjugate hydrolase has also been investigated in terms of activity. Since the generation of transgenic tomato plants is more time consuming than transformation of Arabidopsis, we have created transgenic lines overexpressing IAA amino acid conjugate hydrolases and GH3 genes from different sources and started to analyze these for possible heat stress tolerant phenotypes. Results from work with these transgenic plants will be presented.

MATH-BTB proteins are comprised of 2 domains, MATH (Meprin and TRAF Homology) and BTB (Bric- A-Brac, Tramtrack, Broad Complex). They have been shown to interact with Cul3-based E3 ligase complex which promotes proteasomal degradation of target proteins. The Arabidopsis thaliana genome encodes 6 MATH-BTB genes (AtBPM1-6). Through interaction with members of at least three families of transcription factors (R2R3 MYB family, ethylene response factor/Apetala2 and class I homeobox-leucine zipper transcription factors), BPMs are involved in plant flowering, seed development and abiotic stress response. According to recent findings, BPM proteins are responsible for degradation of transcription factor DREB2A involved in heat- and drought-stress response. To examine physiological roles of BPM1 in heat stress reponse, A. thaliana plants were transformed to obtain transgenic plants overexpressing GFP-tagged BPM1. Homozygous seedlings were exposed to elevated temperature and heat stress followed by whole soluble protein extraction. Anti-GFP monoclonal antibody was used for immunodetection of transgenic GFP-BPM1 protein. In control conditions, recombinant BPM1 exhibited low stability despite high overexpression rates. However, elevated temperatures and heat stress caused significant stabilization of transgenic BPM1. This result could indicate the physiological role of AtBPM1 in response to heat stress in Arabidopsis.

AtBPM1 belongs to the small family of Arabidopsis MATH and BTB domain proteins. A family of MATH-BTB proteins is known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. While BTB domain binds a CUL3 scaffold protein, the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. Tandem affinity purification and yeast two hybrid assay revealed interaction of BPM1 with DMS3 and RDM1, crucial components of RNA-directed DNA methylation pathway (RdDM). Fluorescently tagged BPM1 and its truncated version with deletion of MATH or BTB domain showed that both domains are essential for accumulation of the protein in nucleolus, whilst co-localization results show significant overlap of BPM1 protein with aforementioned RdDM components. Yeast two hybrid assay using truncated BPM1 protein missing MATH or BTB domain revealed that the cullin3-binding BTB domain had higher affinity for RDM1 interaction while both, MATH and BTB domains proved to be equally important for DMS3 interaction. New insights into BPM1 protein and its interaction partners indicate an important, cullin independent function of MATH-BTB protein family in RdDM.

Drought and increased salinity are considered as main abiotic stresses causing the most serious problem for global agriculture effecting plant growth, development and crop yield. Plant responses to osmotic and salinity stress are represented by a number of cellular and molecular processes which are regulated by a complex network of phytohormones. Among them, phytohormone auxin (indole-3-acetic acid, IAA) is the key one mediating plant responses to stress conditions. In order to clarify mechanisms regulating auxin homeostasis in response to osmotic and salinity stress, we have focused on the reversible auxin conjugation as a mechanism of stress adaptation. The role of specific genes of auxin homeostasis in abiotic stress response was addressed, namely functional research was performed on the Arabidopsis thaliana lines that carry mutations in the genes for auxin amidohydrolases (ILR, IAR3 and ILL). Furthermore, the potential to invoke salt and osmotic stress tolerance by over-expression of selected auxin amidohydrolases was evaluated. To screen auxin activity and distribution under stress conditions auxin reporter line (DR5::GFP) was used. Local changes in the auxin levels in the root are connected to the re- adjustment of the auxin homeostasis on the whole plant level in response to salt and osmotic stress.

Grk je autohtona hrvatska sorta vinove loze koja se isključivo uzgaja na otoku Korčuli. Jedna je od rijetkih sorti vinove loze s funkcionalno ženskim cvijetom, što uzrokuje određene probleme u proizvodn ji grožđa uzrokovane smanjenom stopom uspješne oplodnje. Grozd Grka u punoj zrelosti sadrži kombinaciju potpuno razvijenih bobica sa sjemenkama, ali i značajan udio manjih besjemenih bobica, čiji optimalni udio ima pozitivan utjecaj na kvalitetu vina. I mu ški i ženski gametofiti Grka razvijaju se normalno te u zrelosti sadrže dvije spermalne stanice u muškom odnosno jajnu i središnju stanicu u ženskom gametofitu. Glavni razlog umanjene stope oplodnje i posljedični razvoj besjemenih plodova je nevijabilonost peludnog zrna uzrokovana akolporatnom građom vanjske stijenke peludnog zrna kao i izostankom uspješne oplodnje nakon strano - oprašivanja zbog čega dolazi do propadanja sjemenog zametka

One of the major challenges in modern agriculture is overcoming the problem of reduced crop yield due the presence of abiotic stressors. Salinity stress combines two different stress stimuli: toxic effect of sodium and chloride ions and osmotic stress due the occurring dehydration. Plants respond to environmental triggers by complex hormonal crosstalk. In order to investigate the role of one of the major growth promoting substances, indole-3-acetic acid (IAA), different Brassica cultivars: Chinese cabbage (B. rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata) and kale (B. oleracea var. acephala) were exposed to short term 24 h salinity stress (50-200 mM NaCl). Phytohormonal profiling revealed altered levels of stress hormones as well shift in auxins homeostasis which was the most prominent in the most sensitive Chinese cabbage. Presence of salt stressor resulted in higher biosynthetic rate of IAA via activation of indole-3-acetamide pathway. In parallel, the amount of IAA irreversible catabolic forms: 2- oxoindole-3-acetic acid (oxIAA) and indole-3- acetyl-L-glutamate (IAA-Glu) was increased upon salinity too. Higher gene expression levels of auxin-amidohydrolases showed contribution of reversible conjugation process of auxin homeostasis to salinity as well. Beside the perturbations in auxin metabolom, changes in IAA distribution in root were achieved using the A. thaliana DR5rev::GFP auxin reporter lines. Accumulation of GFP signal in vivo and consequently difference in IAA pattern upon 100 mM sodium chloride and 200 mM mannitol treatment was observed by confocal microscope.

DNA methylation is one of several epigenetic mechanisms used by cells to defend the genome against selfish DNA and to control gene expression. DNA methylation and demethylation control genome stability, responses to biotic and abiotic stresses and regulate develo pment. Promoter- methylated genes generally show tissue- specific expression and methylation within promoter regions commonly leads to lower gene expression. Plant DNA methylation occurs at CG, CHG and CHH (an asymmetric site, where H is A, C or T) sequences, each of which has different genetic requirements for the preservation. Nonsymmetrical CHH methylation cannot be sustained by the maintenance and require de novo methylation in each cell cycle through a process called RNA-directed DNA methylation (RdDM) . Two plant specific RNA polymerases, Pol IV and Pol V produce 24 –nucleotide small interfering RNAs and long non-coding RNAs respectively. In th is prosess the DDR complex is required for Pol V- chromatin interaction and transcription, while de novo methylase is targeted to specific locus by nascent Pol V transcript and methylate cytosine in C HH context. It is still not clear how DDR and Pol V are targeted to specific regions in the genome and to what extent RdDM regulate gene expression. Small family of BPM proteins from Arabidopsis has important role in plant flowering, seed development and abiotic stress responses. BPMs directly interact with different transc ription factors and target them for proteasomal degradation. Furthermore, in TAP tag experim ent we identified proteins from DDR complex as BPM1 interactors and suppose that DDR and Pol V could be recruited to promoters by BPMs and sequence specific transcription factors, as occurs with Pol II. The possible role of BPM proteins in navigating RdDM will be presented.

The rules of genetic code are established by aminoacyl-tRNA synthetases (aaRSs), enzymes that covalently link tRNA with its cognate amino acid. Many aaRSs are involved in diverse cellular processes beyond translation by forming protein- protein interactions. However, the studies of plant aaRS assemblies are scarce. Recently, we identified BEN1 as protein interactor of Arabidopsis seryl-tRNA synthetase (SerRS). BEN1 is a putative oxidoreductase involved in metabolism of steroidal plant hormones brassinosteroids. These plant growth-promoting hormones regulate a variety of physiological processes crucial for normal plant growth and development and are also involved in plant stress response. The SerRS:BEN1 complex is the first protein complex of plant aaRSs discovered so far and also one of the rare examples of interaction of aaRSs with enzymes involved in primary or secondary metabolism. To pinpoint regions responsible for interaction, truncated variants of both SerRS and BEN1 were prepared and analyzed using microscale thermophoresis, that enables KD determination. Detailed analysis showed that interaction interface involves SerRS globular catalytic domain and acidic N-terminus of BEN1. Moreover, first 17 amino acids of BEN1 is necessary, but not sufficient for interaction, implying that other parts of BEN1 also participate in interaction with SerRS. Furthermore, using RT-qPCR we observed that expression of BEN1 gene in transgenic plants overexpressing SerRS is slightly lower compared to wild type. To test whether SerRS regulates BEN1 expression in nucleus we determined its localization. Our data show that SerRS is exclusively localized in cytosol, despite its several putative nuclear localization signals. We also investigated the role of SerRS in stress using transgenic plants overexpressing SerRS. Our results show that transgenic seeds germinate earlier and that in most cases transgenic seedlings grow better on various stress media compared to wild type seedlings. This indicates that enhanced expression of SerRS plays a role in the response of Arabidopsis to abiotic stress conditions. Considering that brassinosteroids are also involved in plant stress response we discuss functional importance of SerRS:BEN1 assembly.

It is well known that aaRSs can be involved in diverse cellular functions beyond translation independently or by forming protein-protein interactions. However, the studies of plant aaRS assemblies are scarce. We used high- throughput interactome technologies to identify potential protein interactors of Arabidopsis thaliana SerRS. We generated transgenic plants expressing SerRS-TAP and subsequent TAP-MS analysis revealed several potential interactors, including glutathione S- transferase PM24 and aluminium induced protein AT4g27450. Yeast two hybrid screen indicated interaction with BEN1, protein involved in metabolism of steroidal plant hormones brassinosteroids. These plant growth- promoting hormones regulate a variety of physiological processes crucial for normal plant growth and development and are also involved in plant stress response. The SerRS:BEN1 interaction was confirmed using surface plasmon resonance and microscale thermophoresis (MST), that enable Kd determination. To pinpoint regions responsible for interaction, truncated variants of both SerRS and BEN1 proteins were prepared and analyzed using MST. Kd for complex containing BEN1 and truncated SerRS variant without basic C-terminal extension was similar to Kd of the SerRS:BEN1 complex indicating that basic C- terminus does not participate in interaction. N-terminal tRNA binding domain of SerRS did not form the complex with BEN1. Deletion of N- terminal extension of BEN1 containing six acidic amino acids disrupted the interaction. Therefore we conclude that interaction involves SerRS globular catalytic domain and acidic N- terminus of BEN1. Furthermore, using RT-qPCR we investigated possible correlation of these two proteins on the level of gene expression. We observed slightly lower expression of BEN1 gene in transgenic plants overexpressing SerRS compared to wild type. To test whether SerRS regulates BEN1 expression in nucleus we determined its localization using GFP- localization experiments and immunoblot analysis. Our data show that SerRS is exclusively localized in cytosol, despite its several putative nuclear localization signals. To investigate the role of SerRS in stress, we examined growth of transgenic seedlings overexpressing SerRS on growth media that induce ionic stress, osmotic stress and stress imposed by heavy metals. Our results show that transgenic seeds germinate earlier and that in most cases transgenic seedlings grow better on stress media compared to wild type seedlings. This indicates that enhanced expression of SerRS plays a role in the response of plant to various stress conditions. Considering that brassinosteroids are also involved in plant stress response we propose functional importance of SerRS:BEN1 assembly.

Aminoacyl-tRNA synthetases (aaRS) play significant role in translation process by binding amino acids to their cognate tRNAs. Once the tRNA is charged, a ribosome can transfer the amino acid from the tRNA onto a growing peptide, according to the genetic code. Beyond translation, these enzymes can be involved in diverse cellular functions. Characterization of these non-canonical functions broadens our knowledge in functional proteomics. The studies of aaRS assemblies in plants are scarce, therefore our main scientific goals were to determine and characterize potential protein- protein interacting partners of cytosolic seryl- tRNA synthetase (SerRS) from plant Arabidopsis thaliana. We conducted yeast-two hybrid (Y2H) screen on cDNA libraries followed by DNA sequencing, as well as tandem affinity purification combined with mass spectrometry analysis (TAP-MS). Among several SerRS potential interacting partners revealed by Y2H screen, BEN1, protein potentially involved in metabolism of brassinosteroid hormones was the most promising interacting partner. Interaction of BEN1 and SerRS was also analyzed in vitro using isothermal calorimetry titration (ITC), pull-down and surface plasmon resonance method (SPR). Probably due to the nature of interaction we were not able to retrieve positive results using pull-down assay and ITC, but SPR gave us positive confirmation and information about dissociation constant. To pinpoint regions responsible for protein- protein interaction we prepared shortened variants of both SerRS and BEN1 proteins which will be further subjected to biophysical analysis. Biophysical determination of possible interactions between SerRS and BEN1 variants will give us insight in additional cell functions and physiology of both SerRS and BEN1 proteins. TAP-MS analysis of transgenic plant overexpressing SerRS-TAP construct identified several SerRS potential interacting partners yet to be confirmed in vitro. Revealing plant SerRS interactome has great importance in shedding light on novel functions of SerRS beyond translation.

In Arabidopsis thaliana, the MATH-BTB (BPM) proteins comprise a small family of six members. BPMs act as substrate-binding adaptors for the Cullin3-based ubiquitin E3 ligase, and interect with a broad range of Ethylene response factor/apetala2 transcription factors and with homeodomain-leucine zipper transcription factor ATHB6 affecting fatty acid metabolism and abscisic acid signaling. We showed previously that AtBPM1, localizes predominantly in nucleolus of plant cells indicating a Cullin3 independent function. To further elucidate the molecular function of AtBPM1, we identified AtBPM1 binding and functional partners using tandem affinity purification and mass spectrometry. Different stress-related proteins were identified in AtBPM1 protein complexes such as Catalases CAT2 and CAT3, Nucleoside diphosphate kinase III, Glycine-rich RNA-binding proteins GRP7 and GRP8, MLP-like protein 423, Polyketide cyclase/dehydrase and lipid transport superfamily protein. Moreover, we have identified a set of DNA repair and chromatin remodeling proteins, such as Nucleosome assembly protein NAP1, Tudor-SN proteins, DNA-damage- repair/toleration protein DRT102, WD-40 repeat family protein/beige-related, Chromatin remodeling 34, DNA mismatch repair protein Msh6-1 as well as a known components of RNA-directed DNA methylation, defective in meristem silencing 3 DMS3 and RNA-directed DNA methylation 1 RDM1. Furthermore, direct interaction of AtBPM1 and DMS3 was confirmed by yeast two hybrid and pull down assays and DNA methylation in plants overexpressing AtBPM1 was reduced. These results, for the first time, links MATH-BTB proteins with DNA methylation and related mechanisms.

Aminoacyl-tRNA synthetases (aaRSs) play a critical role in translation by catalyzing the ligation of their cognate amino acids and tRNAs to establish the genetic code. Beside their canonical role in translation, aaRSs participate in various cellular processes, including response to stress conditions. Research on the role of aaRSs in stress-responses in plants is very scarce. One proteomic investigation showed upregulation of Arabidopsis seryl-tRNA synthetase (SerRS) in the early response of A. thaliana cells to cadmium exposure. To further investigate the role of SerRS in the stress, we examined growth of Arabidopsis transgenic seedlings overexpressing SerRS. We tested different stress media that induce ionic stress, osmotic stress and stress imposed by heavy metals. Influence of the plant hormones, abscisic acid and brassinosteroids, was also investigated. Our results show that transgenic seeds germinate earlier and that in most cases transgenic seedlings grow better on stress media compared to wild type seedlings. This confirms that enhanced expression of SerRS plays a role in the response of plant to various stress conditions. Stress related function of SerRS may be linked to its cellular localization. To determine subcellular destination of the SerRS protein and targeting properties of its basic C-terminus we prepared three fusion constructs: GFP-SerRS, SerRS-GFP and GFP-C terminus. All constructs were transiently transformed into heterologous epidermal onion cells and homologous Arabidopsis leaves protoplasts. GFP fluorescence was examined under confocal microscope. For all constructs cytosolic localization was observed. In addition, some onion cells showed accumulation of full-length GFP-SerRS or SerRS–GFP fusion proteins in the nucleus. In Arabidopsis protoplasts nuclear localization was observed only in the case of full-length GFP-SerRS fusion protein. In both onion cells and Arabidopsis protoplasts SerRS C- terminus did not exhibit targeting properties of nuclear localization signals. Because GFP- localization experiments gave ambiguous results we performed immunoblot analysis of cytosolic and nuclear fraction of Arabidopsis leaves. Western blot analysis performed so far indicates cytosolic localization of Arabidopsis SerRS protein.