This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.

Stress-inducible linker histone variant is required for adaptive response of Arabidopsis to complex environmental stress.

Festuca pratensis (meadow fescue) as the most frost-tolerant species within the Lolium–Festuca complex was used as a model for research aimed at identifying the cellular components involved in the cold acclimation (CA) of forage grasses. The work presented here also comprises the first comprehensive proteomic research on CA in a group of monocotyledonous species which are able to withstand winter conditions. Individual F. pratensis plants with contrasting levels of frost tolerance, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on the analysis of leaf protein accumulation before and after 2, 8, and 26 h, and 3, 5, 7, 14, and 21 d of CA, using high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry. The analyses of approximately 800 protein profiles revealed a total of 41 (5.1%) proteins that showed a minimum of a 1.5-fold difference in abundance, at a minimum of one time point of CA for HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analysed plants appeared relatively early during cold acclimation, most often after 26 h (on the 2nd day) of CA and one-half of the differentially accumulated proteins were all parts of the photosynthetic apparatus. Several proteins identified here have been reported to be differentially accumulated during cold conditions for the first time in this paper. The functions of the selected proteins in plant cells and their probable influence on the level of frost tolerance in F. pratensis, are discussed.

Perennial ryegrass (Lolium perenne L.) belongs to the common cultivated grass species in Central and Western Europe. Despite being considered to be susceptible to drought, it is frequently used for forming the turf in urban green areas. In such areas, the water deficit in soil is recognized as one of the most important environmental factors, which can limit plant growth. The basic aim of this work was to explore the mechanisms standing behind the changes in the photosynthetic apparatus performance of two perennial ryegrass turf varieties grown under drought stress using comprehensive in vivo chlorophyll fluorescence signal analyses and plant gas exchange measurements. Drought was applied after eight weeks of sowing by controlling the humidity of the roots ground medium at the levels of 30, 50, and 70% of the field water capacity. Measurements were carried out at four times: 0, 120, and 240 h after drought application and after recovery (refilling water to 70%). We found that the difference between the two tested varieties’ response resulted from a particular re-reduction of P700⁺ (reaction certer of PSI) that was caused by slower electron donation from P680. The difference in the rate of electron flow from Photosystem II (PSII) to PSI was also detected. The application of the combined tools (plants’ photosynthetic efficiency analysis and plant gas exchange measurements) allowed exploring and explaining the specific variety response to drought stress.