Morpho-colorimetric characterization by image analysis to identify diaspores of wild plant species

Digital images of ex situ germplasm stored in the Sardinian Germplasm Bank (BG-SAR) were used for the application of image analysis techniques at the Stazione Sperimentale di Granicoltura per la Sicilia. The analysed accessions refer to 148 taxonomic units belonging to 102 genera and 47 families, typical of the Sardinian flora, and of the Mediterranean basin in general.The images of diaspores were acquired by a flatbed scanner and elaborated with a macro specially developed for the morphometric and colorimetric measurements. This method allowed carrying out a database for the characterization of autochthonous germplasm in entry to the bank and the realization of statistic classifiers for the discrimination of genera and species within the following families: Apiaceae, Boraginaceae, Caryophyllaceae, Cistaceae, Fabaceae and Scrophulariaceae. Such classifiers, based on the linear discriminant analysis (LDA) technique and checked by cross-validation, showed a performance included between 74.3% and 96.4%.In addition, for the genus Astragalus, it was possible to elaborate a classifier able to identify very similar taxa of a species complex, obtaining a performance between 83.7% and 100%. Such analysis proved the validity of the methodology also from the taxonomic point of view.Suggestions for subsequent methodological progress, which could offer applications in other research issues, such as ecological analysis, soil seed bank and archaeological botany are proposed.     

Morphological and functional seed traits of the wild medicinal plant Dioscorea strydomiana,the most threatened yam in the world

• Morphological and functional seed traits have important roles in characterising the species regeneration niche and help to understand the reproductive biology of rare and threatened plants, which can thus support appropriate plant conservation measures.
• Seed morphometric and dispersal kinetics of the critically endangered Dioscorea strydomiana were measured and compared with those of four other Dioscorea species, and seed germination response under constant temperatures (5–35 °C) was compared with that of the congeneric and widespread D. sylvatica.
• Seed mass of D. strydomiana (ca. 14 mg) was twice that of D. sylvatica, but similar to or smaller than the other species examined. Seeds of D. strydomiana have the lowest speed of descent and lowest variability in most of the morphological traits considered, suggesting lower phenotypic plasticity but higher variance in the wing‐loading value. Seeds of D. strydomiana reached maximum germination at 15 °C (ca. 47%), which decreased slightly to ca. 37% at 25 °C and was completely inhibited at 35 °C. D. sylvatica seeds started to germinate at 10 °C (ca. 3%), reached 75–80% germination at 15–20 °C and maximum (ca. 90%) at 25–30 °C. Base temperatures for germination (T_b) were 9.3 and 5.7 °C, for D. strydomiana and D. sylvatica, respectively. Due to the higher germination percentages of D. sylvatica, ceiling and optimum temperatures could also be modelled for this species, suggesting higher sensitivity to high temperature for seeds of D. strydomiana.
• The detected poor seed lot quality of D. strydomiana suggests difficulties in reproduction from seed, highlighting the need for further investigation and conservation actions for this threatened yam species.

     

VGF Peptide Profiles in Type 2 Diabetic Patients’ Plasma and in Obese Mice

To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment.     

Sequential temperature control of multi-phasic dormancy release and germination of Paeonia corsica seeds

Aims The physiological responses during dormancy removal and multi-phasic germination were investigated in seeds of Paeonia corsica (Paeoniaceae).Methods Seeds of P. corsica were incubated in the light at a range of temperatures (10–25 and 25/10°C), without any pre-treatment, after W (3 months at 25°C), C (3 months at 5°C) and W + C (3 months at 25°C followed by 3 months at 5°C) stratification, and a GA 3 treatment (250 mg·l^-1 in the germination substrate). Embryo growth, time from testa to endosperm rupture and radicle emergence were assessed as separate phases. Epicotyl–plumule emergence was evaluated incubating the germinated seeds at 15°C for 2 weeks, at 5 and 25°C for 2 months on agar water before transplanting to the soil substrate at 10, 15 and 20°C and at 15°C for 2 months on the surface agar water with GA 3 .Important findings Embryo growth, testa rupture, endosperm rupture (radicle emergence) and growth of the epicotyl were identified as four sequential steps in seeds of P. corsica. Gibberellic acid alone and warm stratification followed by 15°C promoted embryo growth and subsequent seed germination. Cold stratification induced secondary dormancy, even when applied after warm stratification. After radicle emergence, epicotyl–plumule emergence was delayed for ca. 3 months. Mean time of epicotyl–plumule emergence was positively affected by cold stratification (2 months at 5°C) and GA 3. P. corsica seeds exhibited differential temperature sensitivity for the four sequential steps in the removal of dormancy and germination processes that resulted in the precise and optimal timing of seedling emergence.     

Irradiance stress responses of gas exchange and antioxidant enzyme contents in pariparoba [<Emphasis Type="Italic">Pothomorphe umbellata</Emphasis> (L.) Miq.] plants

We evaluated the growth and development of the medicinal species Pothomorphe umbellata (L.) Miq. under different shade levels (full sun and 30, 50, and 70 % shade, marked as I₁₀₀, I₇₀, I₅₀, and I₃₀, respectively) and their effects on gas exchange and activities of antioxidant enzymes. Photosynthetically active radiation varied from 1 254 μmol m⁻² s⁻¹ at I₁₀₀ to 285 μmol m⁻² s⁻¹ at I₃₀. Stomatal conductance, net photosynthetic rate, and relative chlorophyll (Chl) content were maximal in I₇₀ plants. Plants grown under I₁₀₀ produced leaves with lower Chl content and signs of chlorosis and necrosis. These symptoms indicated Chl degradation induced by the generation of reactive oxygen species. Stress related antioxidant enzyme activities (Mn-SOD, Fe-SOD, and Cu/Zn-SOD) were highest in I₁₀₀ plants, whereas catalase activity was the lowest. Hence P. umbellata is a shade species (sciophyte), a feature that should be considered in reforestation programs or in field plantings for production of medicinal constituents.     

Interchangeable effects of gibberellic acid and temperature on embryo growth, seed germination and epicotyl emergence in Ribes multiflorum ssp. sandalioticum (Grossulariaceae)

Morphophysiological dormancy was investigated in seeds of Ribes multiflorum Kit ex Roem et Schult. ssp. sandalioticum Arrigoni, a rare mountain species endemic to Sardinia (Italy). There were no differences in imbibition rates between intact and scarified seeds, suggesting a lack of physical dormancy, while methylene blue solution (0.5%) highlighted a preferential pathway for solution entrance through the raphe. Embryos were small at seed dispersal, with an initial embryo:seed ratio (E:S) of ca. 0.2 (embryo length, ca. 0.5 mm), whereas the critical E:S ratio for germination was three times longer (ca. 0.6). Gibberellic acid (GA(3), 250 mg · l(-1)) and warm stratification (25 °C for 3 months) followed by low temperature (<15 °C) enhanced embryo growth rate (maximum of ca. 0.04 mm · day(-1) at 10 °C) and subsequent seed germination (radicle emergence; ca. 80% at 10 °C). Low germination occurred at warmer temperatures, and cold stratification (5 °C for 3 months) induced secondary dormancy. After radicle emergence, epicotyl emergence was delayed for ca. 2 months for seeds from three different populations. Mean time of epicotyl emergence was affected by GA(3) . Seeds of this species showed non-deep simple (root) - non-deep simple (epicotyl) morphophysiological dormancy, highlighting a high synchronisation with Mediterranean seasonality in all the investigated populations.     

Anaerobic accumulation of amino acids in rice roots: role of the glutamine synthetase/glutamate synthase cycle

Summary. Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48 h to anaerobic conditions. Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA, inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8 h of anaerobic treatment and, by immunoprecipitation of ³⁵S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during the treatment. These findings indicate that the glutamine synthetase/glutamate synthase cycle play an important role in anaerobic amino acid accumulation. Received April 5, 1999     

Effect of temperature and cold stratification on seed germination of the Mediterranean wild aromatic Clinopodium sandalioticum (Lamiaceae)

A germination study was carried out on seeds of Clinopodium sandalioticum (Bacch. & Brullo) Bacch. & Brullo ex Peruzzi & Conti (Lamiaceae), a wild aromatic plant endemic to Sardinia. Seeds were incubated at a range of constant (5–25°C) and an alternating temperatures regime (25/10°C), with 12 hours of irradiance per day. The results achieved at 10°C were also compared with those obtained after a period of cold stratification at 5°C for three months. Final seed germination ranged from ca. 28% (5°C) to ca. 72% (25/10°C). A base temperature for germination (T_b) of ca. 5°C and a thermal constant for 50% germination (S) of 89.3°Cd were identified and an optimal temperature for germination (T_o) was estimated to be comprised between 20 and 25°C. Cold stratification negatively affected seed viability and germination at 10°C. Although a typical “Mediterranean germination syndrome”, could not be detected for C. sandalioticum seeds, these results were coherent with those previously reported for other Mediterranean Lamiaceae species.