Key papers on stress combination

Shaar-Moshe L., Blumwald E. & Peleg Z. 2017. Unique physiological and transcriptional shifts under combinations of salinity, drought and heat. Plant Physiology, 174: 421-434.

Mittler R. 2006. Abiotic stress, the field environment and stress combination. Trends in Plant Science 11: 15-19.

Mittler R, Blumwald E. 2010. Genetic engineering for modern agriculture: challenges and perspectives. Annual Review of Plant Biology 61: 443-462.

Shaar-Moshe L., Hayouka R., Roessner U., & Peleg Z. 2019. Phenotypic and metabolic plasticity shapes life-history strategies under combinations of abiotic stresses. Plant Direct, 3: 1-13.

Rivero RM, Mestre TC, Mittler R, Rubio F, Garcia-Sanchez F, Martinez V. 2014. The combined effect of salinity and heat reveals a specific physiological, biochemical and molecular response in tomato plants. Plant, Cell & Environment 37: 1059-1073.

Zandalinas SI, Mittler R, Balfagon D, Arbona V, Gomez-Cadenas A. 2018. Plant adaptations to the combination of drought and high temperatures. Physiologia Plantarum 162: 2-12.

Rizhsky L, Liang H, Mittler R. 2002. The combined effect of drought stress and heat shock on gene expression in tobacco. Plant Physiology 130: 1143-1151.

Suzuki N, Rivero RM, Shulaev V, Blumwald E, Mittler R. 2014. Abiotic and biotic stress combinations. New Phytologist 203: 32-43.

coming soon

o   Fingerprinting Antioxidative Activities in Plants (Contributed by Livia Saleh and Christoph Plieth)

o   Metabolic and Proteomic Markers for Oxidative Stress. New Tools.

o   Oxidative stress and antioxidative systems

o   A Comparison of Screening Methods in Brassica napus L.

o   Adventitious root porosity

o   Calculation of nitrogen-use efficiency

o   Handbook of Reference Methods for Plant Analysis, 1998 (classic)

o   Recent developments in fast spectroscopy for plant mineral analysis, 2015

o   Cell Membrane Stability (CMS) by the electro-conductivity method

o   Chlorophyll content reduction under heat stress – see Leaf chlorophyll

o   Chlorophyll Fluorescence (see under General Stress above)

o   Pollen dysfunction under heat stress (see under General Stress above)

o   High-Throughput Screening of Temperature-Sensitive rice grain a-Amylase

o   Heat tolerance phenotyping in the growth chamber – important issues to consider

The method is simple and straightforward as described by Wang et al. (Aust. J. Agric. Res. 57(1) 89–100, 2006). A minimal extract from that paper is presented below:

From Wang et al. (Aust. J. Agric. Res. 57(1) 89–100, 2006):

Andrzej Anio, of the Plant Breeding and Acclimatization Institute, Radzikow, 05-850 Blonie, Poland, also published the details of the method in ‘Plant Breeding News’ (online):

The test used in our experiments is based on the assumption that inhibition of plant growth by Al is not observed before root systems develop. Since inhibition of root elongation is the first visible symptom of Al injury, direct reference to this process in selection seems to be a reasonable approach.

The pulse test is based on exposure of roots to short Al shock after which the effect on root elongation is recorded. In the method used in our laboratory, the Al pulse principle of MOORE ET AL.1976 was combined with the staining technique developed by POLLE ET ALL 1978. Seeds were sterilized with 0.1%Hg₂ Cl₂ aqueous solution for 10 min., rinsed thoroughly with water, and germinated overnight on filter paper in Petri dishes. Sprouted seeds were sown the next day on polyethylene net fixed in lucite frames. Styrofoam blocks were attached to the frames with rubber bands and floated on the surface of a vigorously aerated nutrient solution. Containers with nutrient solution were placed in water bath at 25^oC under continuous light (12 w/m). Nutrient solution of the following composition was used: 0.4 mom calcium chloride, 0.65 mom potassium nitrate, 0.25 mom magnesium chloride, 0.01 mom ammonium sulfate and 0.04 mom ammonium nitrate. Four day-o1d seedlings were transferred to the same nutrient medium supplied with Al, in the form of aluminum chloride, at concentration indicated in the experiments. After 24 hours of incubation in the medium containing Al, seedlings were thoroughly washed for 2-3 min. in running water and transferred to nutrient solution without Al for 48 h. Seedlings were removed from nutrient solution, again washed with tap water, and stained with 0.1 % aqueous solution of Eriochrome cyanine R for 10 min. The excess dye was washed after staining with tap water. The root regrowth after Al shock (or additional root growth) was easily assessed. Seedlings with apical meristems, damaged by a given Al pulse, had intensively stained root tips while those not damaged by Al had a stained section of root followed by white root tip which developed after Al shock. The dye is nontoxic to roots at the concentration used and at the time stain was applied. During all stages of growth, and particularly during Al treatment, nutrient solution was maintained at pH 4.5, adjusted with 0.1 M HCl. At the ratio of approximately 20 ml of nutrient solution per seedling, changes of pH of the medium were minimal during 24 h of Al treatment. Seedlings after the test were still viable and could be transplanted for seed increase. Aluminum concentration in nutrient solution causing irreversible damage to root apical meristem during pulse treatment is a measure of tolerance of tested genotype.

The described test was used for screening parental cultivars and hybrid populations. The results correspond very well with cultivars of known Al resistance. Selected seedlings were transplanted to the field for seed increase or further crossing. The test gives reproducible results provided that proper conditions (temperature, pH, Al concentration, time of exposure to Al) are controlled.

This test was used successfully in our program directed at development of winter wheat strains with introduced Al-tolerance from Brazilian, extensive spring cultivar BH 1146. We can now offer to the wheat breeders a set of strains with agronomic characters at the level of top cultivars on the list and Al-tolerance similar to BH 1146.

Several major genes control Al tolerance in hexaploid wheat and the character is dominant and heritable, therefore, selection for Al tolerance in wheat would be effective.

The described screening test does not allow the discrimination between Al-tolerant homozygotes and heterozygotes. Moreover, Al-tolerance in hexaploid wheat is a genetically complex character and sensitive segregants were observed in many generations after cross. This obstacle was solved by development of DH lines in F₃ /F₄ generations.

References

Moore D P, Kronstad W E and Metzger R J 1976. Screening wheat for aluminum tolerance. In: Plant Adaptation to Mineral Stress in Problem Soils. Ed. M J Wright. Cornell Univ Agric. Exp. Sta.,Ithaca,NY.

Polle E, Konzak A F and Kittric J A 1978 Visual detection of aluminum tolerance levels in wheat by hematoxylin staining of seedling roots. Crop Sci. 18, 823-827.

Aluminum toxicity tolerance is largely expressed by the exclusion of aluminum from roots or its binding, thereby avoiding absorption and toxification. For details see ‘Mineral Toxicity’ under our ‘The Stresses’ page. Polle et al. (1978) established a method to evaluate for this tolerance mechanism in cereal crop plants by hematoxylin root staining. Hematoxylin binds aluminum to produce a purple complex. The absence of the color in root tips treated by hematoxylin indicates that these plants either exclude or bind aluminum in complexes that are unavailable to hematoxylin. Since that publication, the method has been used extensively, with or without slight modifications, for quick evaluation of and screening for aluminum tolerance.

A typical protocol begins by growing germinated seedlings in nutrient solution (preferably aerated) set to pH 4.5. After 2-3 days the nutrient solution is replaced with an identical one which also includes 50 mM AlCl₃. Other concentrations can be used depending on the specific study. A preliminary test with several aluminum concentrations is recommended before a large scale screening is embarked upon. Use of standard tolerant and susceptible cultivars is important for scaling the results.

After 24h of exposure to aluminum the seedlings are washed with de-ionized water for 30 min (with several changes of water) after which the roots are exposed for 30 min to a solution of hematoxylin. Following staining the roots are washed again for 30 minutes. The different levels of root tip staining (see example in Fig.1) can be visually scored and/or photographed.

The hematoxylin stain is prepared by placing 0.2 g of hematoxylin and 0.02 g of KI0₃ in 100 ml of water and keeping the solution overnight to dissolve the hematoxylin.

Fig.1. Differential hematoxylin staining of root tips in different barley cultivars. Left– tolerant; right-susceptible. (from Tang et al., 2000, Crop Sci.40:778-782) 

References

Polle E., Konzak C.F. and Kittrick J.A. 1978. Visual detection of aluminum tolerance levels in wheat by hematoxylin staining. Crop Sci 18: 823-827

Tang Y., Sorrells M.E., Kochian L.V. and Garvin D.F. 2000. Identification of RFLP markers linked to the barley aluminum tolerance gene Alp   Crop Sci.40:778-782.