Foteini Kolyva*(1), Stratakis E. (2), Meletiou-Christou M.S. (1), Rhizopoulou S. (1)

(1) Department of Botany, Faculty of Biology, National and Kapodistrian University of Athens,Panepistimiopolis, 15784 Athens, (2) IESL, FORTH, Heraklio, Crete

*fotinikoliva@biol.uoa.gr

The impact of climatic change in viticulture is expected to be severe in the Mediterranean area in the future [1, 2]. The scope of this study is the evaluation of the leaf functional and optical properties of grapevine (Vitis vinifera L.) cultivar (cv.) Assyrtiko and its response to abiotic stress conditions (temperature and water deficiency) caused by climatic change. Plants of grapevine cv. Assyrtiko were placed in a growth chamber in the Botany Department of the National and Kapodistrian University of Athens, Greece and four treatments were applied; temperature (ambient versus ambient +2^oC) and water availability (full irrigation versus cyclic drought). The accumulation of photosynthetic pigments (chlorophylls a and b and carotenoids) was determined as well as the leaf area, dry weight and specific leaf area in expanding and fully expanded leaves of the treated plants. Using a UV/VIS spectrophotometer (Perkin Elmer Lambda-950), equipped with an integrating sphere [3], the reflectance (R) and the transmittance (T), between 250 and 2500 nm wavelength [4,5] were measured in situ, for both adaxial and abaxial leaf surfaces of the grapevine var. Assyrtiko. Assessing the above data was found that combined drought with elevated temperature, reduces chlorophyll accumulation. The selected data along with the parameters of the abiotic environment, were tested for model simulation purposes that will help the mitigation of the impact of climatic change on the (Vitis vinifera L.) cultivar (cv.) Assyrtiko in Greece.

[1] Gauquelin T, et al. Regional Environmental Change 18(3), 623-636 (2018)

[2] Del Pozo A, et al. Sustainability 11(10), 2769 (2019)

[3] Stratakis E, et al. Progress in Quantum Electronics 33(5), 127-163 (2009)

[4] Jacquemoud S, et al. Remote Sensing of Environment 56(3), 194-202 (1996)

[5] Jacquemoud S, Ustin S. Leaf optical properties. Cambridge University Press (2019)

Abiotic stress adversely affects crop production, causing yield reductions in important crops, including tomato (Solanum lycopersicum). Among different abiotic stresses, drought is considered to be the most critical one since limited water availability negatively impacts plants growth and development, especially in arid or semi-arid areas. The aim of this study was to understand how biostimulants may interact with critical physiological response mechanisms in tomato under limited water availability and to define strategies to improve tomato performances under drought stress. We investigated physiological responses of the tomato genotype E42 grown in open field under control condition (100% irrigation) and limited water availability (50% irrigation) and treated or not with a novel plant-based biostimulant named CycoFlow (Agriges). Under limited water availability plants treated with the biostimulant showed a reduction in osmotic pressure (-22.50% vs non treated plants). The biostimulant treatment enhanced plant growth (biomass) under control (+ 99.02% vs non treated plants) and stress conditions (+296.63% vs non treated plants). Also, biostimulant treated plants had higher pollen viability (+ 50.94% vs non treated plants) and higher yield per plant (+ 563.90% vs non treated plants) under limited water availability. The treatment with the biostimulant had also an effect on antioxidants and pigments content, both in leaves and fruits. In particular, the content of phenols increased under limited water availability (+ 11.83% vs non treated plants), while the ascorbic acid content decreased (- 7.33% non treated plants). Altogether, these results indicate that the application of the biostimulant CycoFlow to tomato plants improved plant performances under limited water availability.

Changing climatic conditions played a major role in plants evolution and could modify the composition, structure and functionality of native plant communities, favoring invasive species. Studies conducted in south-eastern Australia have reported Pittosporum undulatum, a native tree, to be an aggressive invader of Eucalyptus forests. We tested the hypothesis that its negative impact on floristic diversity is due to the release of allelopathic compounds inhibiting the germination and growth of other plants. Thus, we compared the germination of Pittosporum undulatum, Eucalyptus ovata and lettuce on different substrates. Seeds were watered with leachates made from fresh P. undulatum leaves, litter collected from underneath P. undulatum or Eucalyptus trees, or distilled water.
P. undulatum seeds germinated more slowly than the other two species, however showed faster growth rates. E. ovata mortality rates seedlings were very high immediately after germination. While there were no significant treatment effects on germination rates, there were differences in morphology. The root system, in lettuce and E. ovata, was short and damaged when watered with fresh P. undulatum leaves extract.
Additionally, we investigated the possible emission of specific Biogenic Volatile Organic Compounds (BVOCs) which could have ecological functions or increase P. undulatum stress resistance. Using Solid Phase Microextraction (SPME) fibers, BVOCs were collected and analysed using Gas chromatography-mass spectrometry (GC-MS) to compare the emissions in eucalypt forests with and without P. undulatum. The main difference between the two sites was the higher D-limonene and α-pinene aerial concentration in P. undulatum infested sites.
Our results suggest that the high invasiveness of P. undulatum is mainly related to morphological and physiological characteristics rather than to allelopathic compounds emitted by this species. Additionally, the greater emission of D-limonene and α-pinene from P. undulatum compared to eucalypts could increase the resistance to abiotic stresses, such as drought or ozone.

Pistacia lentiscus L. leaves are used in several applications, thanks to their polyphenolic abundance. Different methods are used for the extraction of these compounds. We aimed to optimize the ultrasond-assisted extraction (UAE) of P. lentiscus to obtain higher amounts of polyphenols in a greener way. A Box-Behnken design using temperature (30-50 °C), solvent volume (15-30 mL) and ethanol fraction (30-50 %) was conducted. Galloyl and myricetin derivatives were the most abundant compounds detected (HPLC-DAD-MS analysis). The regression analyses of total polyphenol (TPC), total tannins (TTC), total flavonoids (TFC) and myricitrin contents (MYC) showed good fit of the models. The maximum yields of TPC (51.3 ± 1.8 mg g^-1 DW) and TTC (40.2 ± 1.4 mg g^-1 DW) are obtained using 18 mL of 40 % ethanol at 50 °C. For flavonoids, an extraction with 20 mL of 50 % ethanol at 50 °C predicts the highest content (10.2 ± 0.8 mg g^-1 DW), while 15 mL of 30 % ethanol at 30 ° C results in the maximum of MYC (2.6±0.19 mg g^-1 DW). This greener process decreased in 25 % the percentage of ethanol and in half the time of the usual ethanolic extraction. Therefore, these UAE conditions can be applied to obtain polyphenolic enriched extracts from P. lentiscus leaves, which can be further employed for several industrial purposes.

Mexico is a megadiverse country, with a highh quantity of unique plant species; with different uses and applications, such as bactericidal, fungicidal, insecticidal, and recently nutrimental. The content of phytochemicals and the impact of them in the animal and human health, has been make them a target for biotechnological improvement. At the región Bajio in Mexico, have been identified several plants associated with ecological, medical and industrial potential, but also asocciated to the traditions of the population whit no real utility. The work in this Project includes the development of systems for the culture for production of secondary metabolites (in vitro root tissue culture) and the miRNA expresión analysis in order to find the molecules asocciated to metabolites production. In this study we include two plants: marigold (Tagetes erecta), which the genes associated to lutein production had been identified in flower development, systems for cell culture and plant transfromation has been developed, but no systems for in vitro root culture. To now there are not studies related to miRNA expresión and association to these molecules to secondary metabolites. In Heliopsis longipes, several metodologies had been developed for the isolation of afinin and its uses in agriculture, medicine and recently as analgesic activities in some other metabolites. First, a root tissue culture was established for both of the plants (marigold and Heliopsis), using a combination of auxins (2,4-D, IAA, IBA) in a cinetical assay, as the base for manipulation; differences in the root architecture were determined mainly in the time of production and root architecture. In the molecular analysis four miRNAs were found differentially expressed and associated to secondary metabolites production (miR146, miR164, miR168, miR171). The reordering of miRNAs synthesis and the targets was analysed and is associated to the secondary metabolites production in order to establish a system for the in vitro induction of metabolites.

Increase in demand for meat, milk and their products is expected to escalate considerably in coming years due to our ever-expanding population. While increase in forage crop production will therefore be a necessity to meet demand, our ability to attain high levels of forage crop productivity is likely to be constrained typical environmental pressures such as drought and salinity due to climate change. Alfalfa (Medicago sativa L.) is one of the world’s most widely grown forage species with a cropping area of over 30 million hectares worldwide. As such, there is a critical need to exploit advanced molecular breeding technologies in this species with the aim of rapidly developing alfalfa cultivars with improved biomass, as well as resiliency to various types of abiotic stress. It has been shown previously that the RNAi-mediated down-regulation of the miRNA156 target gene, SQUAMOSA PROMOTER-BINDING-LIKE8 (MsSPL8), enhances biomass production, as well as drought and salinity tolerance, in alfalfa. However, due to negative public perception and regulatory constraints surrounding the use of transgenic crops, it remains a challenge to implement such a crop in growers’ fields. CRISPR/Cas9-based genome editing provides an alternative breeding tool that yields germplasm bearing a mutation that is fundamentally identical to those achieved using conventional breeding approaches such as chemical mutagenesis, and the resulting plants can be made transgene-free in a straightforward manner. In this study, we successfully targeted MsSPL8 alleles using this technology in alfalfa, and isolated genotypes with mutations in approximately 25%, 50% and 75% of MsSPL8 alleles, respectively, in this tetraploid species. Furthermore, enhanced drought and salinity resistance, along with distinct morphological alterations including early flowering and reductions in internode length, were noted in the first generation of edited genotypes, which suggests that CRISPR/Cas9 can provide an effective breeding tool in alfalfa.

Alfalfa (Medicago sativa L.) is a perennial legume esteemed for its yield, adaptability and superior nutritional quality as a forage crop. However, alfalfa production is often impacted by various environmental challenges such as drought and poor drainage throughout the growing season, which leads to a decline in farmer’s profitability. These factors are anticipated to become more problematic in the coming years due to global warming scenarios, and as such, there is a need for the development of alfalfa cultivars with enhanced abiotic stress resilience. In this study, five gene homologs (CBF2, ACBP3, TAC1, FAO3 and HB2) negatively regulating various abiotic stresses in other closely related crop species were identified in alfalfa, and RNAi genotypes exhibiting down-regulation of each gene, respectively, were generated. The RNAi genotypes were subjected to drought and flooding treatments, respectively, to assess their responses to abiotic stresses. Preliminary results demonstrated that alfalfa genotypes with reduced expression of TAC1 exhibited increased tolerance to drought, while the down-regulation of ACBP3 and HB2 in alfalfa led to enhanced tolerance to flooding. Further experiments are underway to unravel the mechanisms driving increased abiotic stress tolerance in these genotypes. Our aim is to use the knowledge gained in this study to produce transgene-free highly adaptable alfalfa germplasm using advanced molecular breeding platforms such as genome editing via CRISPR/Cas, which could reduce production costs and enhance biomass production by minimizing forage crop losses under extreme weather conditions.

Commiphora wightii is an important medicinal tree of arid and semi-arid regions of India. It is commonly known as Guggal and belongs to family Burseraceae. It is dioecious in nature and male plants are extremely rare. Female plants produce seeds through apomixis. The slow growing nature, low seed viability and germination, excessive tapping for oleogum resin (known as guggul) put this plant under critically endangered categories in IUCN red data book. It is also reported earlier that this plant produces two types of seeds viz. black seed with higher viability and white seed with no or very poor viability. Therefore, Present study was carried out with the aim to identify the right mature seed harvesting period with higher viable seeds ratio which will be helpful in raising nursery and large-scale plantation of Guggul. The mature seeds were collected from 647 guggal plants (10 years old) from Deesa, Gujarat, India in summer (July, 2017) and winter (November and December, 2017). There is no significant difference in percentage of plants bearing mature seeds but number of mature seeds per seed producing plant was significantly higher in winter (72.51 seeds) as compared with summer (10.19 seeds). The proportion of black seed in summer and winter was almost opposite i.e. in winter 70.9% seeds were black whereas in summer only 30% black seeds were found. Seed germination data revealed that black seeds collected in winter showed higher seed germination (13.6%) than summer (2.1%). White seed collected in summer failed to germinate whereas very low seed germination was observed in winter (1.2%).

Papadopoulou S. *(1), Stratakis E. (2), Meletiou-Christou M.S. (1), Rhizopoulou S. (1)

(1) Department of Botany, Faculty of Biology, National and Kapodistrian University of Athens,Panepistimiopolis, 15784 Athens, (2) IESL, FORTH, Heraklio, Crete

*sopapad@biol.uoa.gr

The optical properties and ecophysiological parameters of carob leaves, Ceratonia siliqua L., expanded in more and less polluted habitats [3], were compared, in order to evaluate the effect of air quality in leaf development [2]. The accumulation of pigments (chlorophylls a and b, and carotenoids) and specific leaf area (SLA, cm² g^-1) were seasonally determined during leaf development (i.e. in nine successively grown leaves along shoots). Leaf transmittance (T) and reflectance (R) spectra for both adaxial and abaxial leaf surfaces were measured between 250 and 2500 nm wavelengths, using a UV-VIS spectrophotometer and leaf absorbance [(Abs = 100 – (R + T)] is used to assess the effect of environmental quality of the two contrasting habitats in Athens, on carob leaf physiology. An increase, in the studied leaf parameters, was observed, for carob trees grown in the urban site. There was an increase in SLA from spring to late summer and a decrease in late autumn. Leaves of the less polluted site, regardless of the developmental stage exhibited greater water absorption, while the adaxial surface absorbed more radiation in both categories of plants. It seems likely that differences in optical properties and pigment accumulation have important implications for model simulation purposes [4] and may be used for air pollution biomonitoring [1].

[1] M.S. Meletiou-Christou, et al. Global NEST Journal, 13, 395-404 (2011)

[2] Zadeh AR et al. Environmental pollution 178, 7-14 (2013)

[3] Brackx et al. Environmental Pollution 220, 159-167 (2016)

[4] Féret, J-B. et al. Remote Sensing of Environment 193, 204-215 (2017)

Licorice (Glycyrrhiza uralensis Fish.) is considered as salt and drought tolerant leguminous plant and is thus used to rehabilitate abandoned saline lands. We hypothesized that the biochar amendment into the soil might alleviate salt stress in licorice by improving its plant growth, nutrient acquisition, and root system. The present study was designed to determine the effect of biochar on licorice plant growth, acquisition of C (carbon), nitrogen (N), and phosphorus (P) and on soil fluorescein diacetate (FDA) and enzyme activities under saline soil condition. Pyrolysis char from maize (MBC, 600oC) at 2, 4, and 6 % concentrations were used for pot experiments. The shoot and/or root biomass of licorice grown in soil amended with 2 and 4% MBC under non-saline and saline conditions was increased. Furthermore, increased nodule numbers of licorice grown at 4% MBC amendment was observed under non-saline and saline conditions. The root architectural traits, like root length, surface area, project area, and root volume, as well as nodulation traits, were also significantly modified by biochar application at both 2 and 4% concentrations. The concentrations of N and K in plant tissue were increased under 2 and 4% MBC amendment compared to plants grown without biochar application. Moreover, soil amended with biochar showed a positive effect on soil FDA activity, proteases, acidic phosphomonoesterases under saline soil conditions compared to the control soil. This study demonstrated the beneficial effects of biochar from maize on growth and nutrient uptake of licorice by improving the nodule formation, root architecture, and soil biological activity in saline soil conditions.