Marcin Rapacz

Reference genes in real-time PCR

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.

Molecular mechanisms underlying frost tolerance in perennial grasses adapted to cold climates

We review recent progress in understanding cold and freezing stress responses in forage grass species, notably Lolium and Festuca species. The chromosomal positions of important frost tolerance and winter survival QTLs on Festuca and Lolium chromosomes 4 and 5 are most likely orthologs of QTLs on Triticeae chromosome 5 which correspond to a cluster of CBF-genes and the major vernalization gene. Gene expression and protein accumulation analyses after cold acclimation shed light on general responses to cold stress. These responses involve modulation of transcription levels of genes encoding proteins involved in cell signalling, cellular transport and proteins associated with the cell membrane. Also, abundance levels of proteins directly involved in photosynthesis were found to be different between genotypes of differing frost tolerance levels, stressing the importance of the link between the function of the photosynthetic apparatus under cold stress and frost tolerance levels. The significance of the ability to undergo photosynthetic acclimation and avoid photoinhibition is also evident from numerous studies in forage grasses. Other interesting candidate mechanisms for freezing tolerance in forage grasses are molecular responses to cold stress which have evolved after the divergence of temperate grasses. This includes metabolic machinery for synthesis of fructans and novel ice-binding proteins.

Identification of leaf proteins differentially accumulated during cold acclimation between Festuca pratensis plants with distinct levels of frost tolerance

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.

GISH/FISH mapping of genes for freezing tolerance transferred from Festuca pratensis to Lolium multiflorum

The first backcross breeding programme for the transfer of freezing-tolerance genes from winter hardy Festuca pratensis to winter-sensitive Lolium multiflorum is described. A partly fertile, triploid F1 hybrid F. pratensis (2n=2x=14) × L. multiflorum (2n=4x=28) was employed initially, and after two backcrosses to L. multiflorum (2x) a total of 242 backcross two (BC2) plants were generated. Genomic in situ hybridisation (GISH) was performed on 61 BC2 plants selected for their good growth and winter survival characters in the spring following one Polish winter (2000–2001). Among the winter survivors, diploid chromosome numbers were present in 80% of plants. An appropriate single Festuca introgression in an otherwise undisturbed Lolium genome could provide increased freezing tolerance without compromise to the good growth and plant vigour found in Lolium. Among all the diploids, a total of 20 individuals were identified, each with a single F. pratensis chromosome segment. Another diploid plant contained 13 Lolium chromosomes and a large metacentric F. pratensis chromosome, identified as chromosome 4, with two large distal Lolium introgressions on each chromosome arm. Three of the diploid BC2, including the genotype with Festuca chromosome 4 DNA sequences, were found to have freezing tolerance in excess of that of L. multiflorum, and in one case in excess of the F. pratensis used as control. A detailed cytological analysis combining GISH and fluorescence in situ hybridisation analyses with rDNA probes revealed that the other two freezing-tolerant genotypes carried a Festuca chromosome segment at the same terminal location on the non-satellite arm of Lolium chromosome 2.

Overwintering of herbaceous plants in a changing climate. Still more questions than answers.

The increase in surface temperature of the Earth indicates a lower risk of exposure for temperate grassland and crop to extremely low temperatures. However, the risk of low winter survival rate, especially in higher latitudes may not be smaller, due to complex interactions among different environmental factors. For example, the frequency, degree and length of extreme winter warming events, leading to snowmelt during winter increased, affecting the risks of anoxia, ice encasement and freezing of plants not covered with snow. Future climate projections suggest that cold acclimation will occur later in autumn, under shorter photoperiod and lower light intensity, which may affect the energy partitioning between the elongation growth, accumulation of organic reserves and cold acclimation. Rising CO2 levels may also disturb the cold acclimation process. Predicting problems with winter pathogens is also very complex, because climate change may greatly influence the pathogen population and because the plant resistance to these pathogens is increased by cold acclimation. All these factors, often with contradictory effects on winter survival, make plant overwintering viability under future climates an open question. Close cooperation between climatologists, ecologists, plant physiologists, geneticists and plant breeders is strongly required to predict and prevent possible problems.

Drought-induced changes in the actin cytoskeleton of barley (Hordeum vulgare L.) leaves

Plants have developed different strategies to adapt to various stress conditions including drought. In the present study the drought-induced changes in the actin filament (AFs) network was studied, for the first time, in two barley cultivars of contrasting drought tolerance level. Detached leaves of drought-tolerant (cv. ‘CAM/B1/CI’) and drought-susceptible (cv. ‘Maresi’) cultivars were dried under controlled conditions. The water relations as well as the transcript accumulation of actin (ACT11), actin depolymerization factor (ADF1) and dehydrine (HVA1) encoding genes were studied using qRT-PCR. Quantitative (the relative fluorescence index; RFI) and qualitative drought-induced changes in AF cytoskeleton were observed following staining with phalloidin. It was noticed that tolerant cultivar was characterized with relative water content decreased during drought treatment which was accompanied by increase in HVA1 expression together with decrease in ACT11 and ADF1 transcripts accumulation induced by drought. In drought-susceptible cultivar the expressions of both ACT11 and ADF1 were slightly lower than those in the control. Drought triggered an extensive AF cytoskeleton reorganization within different types of leaf-blade cells. Remarkable changes in AF configuration and its increased amount (fluorescence intensity) were observed mainly in drought-tolerant cultivar. In addition, drought-induced changes in AFs were closely associated with chloroplasts. Those AFs probably controlled drought-induced intracellular chloroplast positioning in mesophyll. Based on the results obtained in the present study, the possible role of AF rearrangements in drought response is discussed.

The Effects of Cold Acclimation on Photosynthetic Apparatus and the Expression of COR14b in Four Genotypes of Barley (Hordeum vulgare) Contrasting in their Tolerance to Freezing and High-light Treatment in Cold Conditions

BACKGROUND AND AIMS Cold acclimation modifies the balance of the energy absorbed and metabolized in the dark processes of photosynthesis, which may affect the expression of cold-regulated (COR) genes. At the same time, a gradual acclimation to the relatively high light conditions is observed, thereby minimizing the potential for photo-oxidative damage. As a result, the resistance to photoinhibition in the cold has often been identified as a trait closely related to freezing tolerance. Using four barley genotypes that differentially express both traits, the effect of cold acclimation on freezing tolerance and high-light tolerance was studied together with the expression of COR14b, one of the best-characterized barley COR genes. METHODS Plants were cold acclimated for 2 weeks at 2 degrees C. Freezing tolerance was studied by means of electrolyte leakage. Changes in photosynthetic apparatus and high-light tolerance were monitored by means of chlorophyll fluorescence. Accumulation of COR14b and some proteins important in photosynthetic acclimation to cold were studied with western analysis. COR14b transcript accumulation during cold acclimation was assessed with real-time PCR. KEY RESULTS Cold acclimation increased both freezing tolerance and high-light tolerance, especially when plants were treated with high light after non-lethal freezing. In all plants, cold acclimation triggered the increase in photosynthetic capacity during high-light treatment. In two plants that were characterized by higher high-light tolerance but lower freezing tolerance, higher accumulation of COR14b transcript and protein was observed after 7 d and 14 d of cold acclimation, while a higher transient induction of COR14b expression was observed in freezing-tolerant plants during the first day of cold acclimation. High-light tolerant plants were also characterized with a higher level of PsbS accumulation and more efficient dissipation of excess light energy. CONCLUSIONS Accumulation of COR14b in barley seems to be important for resistance to combined freezing and high-light tolerance, but not for freezing tolerance per se.

Frost resistance and cold acclimation abilities of spring-type oilseed rape

Spring forms of oilseed rape are perceived as having low frost resistance. This is consistent with some field observations and laboratory studies in which spring and winter forms showed different extents of metabolic changes during cold acclimation. This is somewhat surprising, since rapeseed cultivars are classified as winter or spring types based on their requirements for vernalization to flower, not on their levels of frost resistance. This discrepancy may be due to the limited abilities of spring-type rape, in commonly used growth conditions, to cease growth and, simultaneously, maintain a high photosynthetic activity during cold acclimation, which is considered necessary for proper hardening. This study was conducted to determine if spring oilseed rape could acclimate to the level observed in winter-type plants after modification of growth and photosynthetic characteristics induced by prehardening. After prehardening spring cultivars of oilseed rape showed frost resistance comparable with winter-type plants and their response to prehardening was even stronger than that in winter-type plants. It is caused by the exceptionally low cold acclimation abilities of non-prehardened spring forms. No differences were noted between the frost resistances of callus tissue derived from winter and spring-type cultivars.

Differences in leaf proteome response to cold acclimation between Lolium perenne plants with distinct levels of frost tolerance

Perennial ryegrass (Lolium perenne) is a high quality forage and turf grass mainly due to its excellent nutritive values and rapid establishment rate. However, this species has limited ability to perform in harsh winter climates. Though winter hardiness is a complex trait, it is commonly agreed that frost tolerance (FT) is its main component. Species growing in temperate regions can acquire FT through exposure to low, non-lethal temperatures, a phenomenon known as cold acclimation (CA). The research on molecular basis of FT has been performed on the model plants, but they are not well adapted to extreme winter climates. Thus, the mechanisms of cell response to low temperature in winter crops and agronomically important perennial grasses have yet to be revealed. Here, two L. perenne plants with contrasting levels of FT, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on analyses of leaf protein accumulation before and after 2, 8, 26 h, and 3, 5, 7, 14 and 21 days of CA, using a 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 (MS). Analyses of 580 protein profiles revealed a total of 42 (7.2%) spots that showed at a minimum of 1.5-fold differences in protein abundance, at a minimum of at one time point of CA between HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analyzed plants appeared most often on the 5th (18 proteins) and the 7th (19 proteins) day of CA. The proteins derived from 35 (83.3%) spots were successfully identified by the use of MS and chloroplast proteins were shown to be the major group selected as differentially accumulated during CA. The functions of the identified proteins and their probable influence on the level of FT in L. perenne are discussed.

A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis

Stress-inducible linker histone variant is required for adaptive response of Arabidopsis to complex environmental stress. Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass of minor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stress-inducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants.