Kumud B. Mishra

Engineered drought tolerance in tomato plants is reflected in chlorophyll fluorescence emission

Drought stress is one of the most important factors that limit crop productivity worldwide. In order to obtain tomato plants with enhanced drought tolerance, we inserted the transcription factor gene ATHB-7 into the tomato genome. This gene was demonstrated earlier to be up-regulated during drought stress in Arabidopsis thaliana thus acting as a negative regulator of growth. We compared the performance of wild type and transgenic tomato line DTL-20, carrying ATHB-7 gene, under well-irrigated and water limited conditions. We found that transgenic plants had reduced stomatal density and stomatal pore size and exhibited an enhanced resistance to soil water deficit. We used the transgenic plants to investigate the potential of chlorophyll fluorescence to report drought tolerance in a simulated high-throughput screening procedure. Wild type and transgenic tomato plants were exposed to drought stress lasting 18 days. The stress was then terminated by rehydration after which recovery was studied for another 2 days. Plant growth, leaf water potential, and chlorophyll fluorescence were measured during the entire experimental period. We found that water potential in wild type and drought tolerant transgenic plants diverged around day 11 of induced drought stress. The chlorophyll fluorescence parameters: the non-photochemical quenching, effective quantum efficiency of PSII, and the maximum quantum yield of PSII photochemistry yielded a good contrast between wild type and transgenic plants from day 7, day 12, and day 14 of induced stress, respectively. We propose that chlorophyll fluorescence emission reports well on the level of water stress and, thus, can be used to identify elevated drought tolerance in high-throughput screens for selection of resistant genotypes.

Evidence for a role of raffinose in stabilizing photosystem II during freeze–thaw cycles

A role of non-reducing sugars like sucrose and raffinose in the protection of plant cells against damage during freezing has been proposed for many species, but reports on physiological effects are conflicting. Non-aqueous fractionation of mesophyll cell compartments in Arabidopsis thaliana was used to show that sucrose and raffinose accumulate in plastids during low temperatures, pointing to a physiological role in protecting the photosynthetic apparatus. Comparing a previously described raffinose synthase (RS) mutant of A. thaliana with its corresponding wild type, accession Col-0, revealed that a lack of raffinose has no effect on electrolyte leakage from leaf cells after freeze–thaw cycles, supporting that raffinose is not essential for protecting the plasma membrane. However, in situ chlorophyll fluorescence showed that maximum quantum yield of PS II photochemistry (Fv/Fm) and other fluorescence parameters of cold acclimated leaves subjected to freeze–thaw cycles were significantly lower in the raffinose synthase mutant than in the corresponding wild type, indicating that raffinose is involved in stabilizing PS II of cold acclimated leaf cells against damage during freezing.

Chlorophyll fluorescence emission as a reporter on cold tolerance in Arabidopsis thaliana accessions.

Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around -15°C. This, together with the method’s easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method.

Plant phenotyping: a perspective

Sustainable agriculture for feeding increasing population is a foremost global challenge. The “green revolution” based crop productivity has done wonders in the past, but it has limits, and, thus, we are compelled to look for new avenues to increase productivity of important crops. Plant phenomics is emerging as a promising area in which many imaging sensors developed in the past are being tested for mapping of genetic information expressed within plant phenotypes, and the integrated use of these sensors may help speed-up unraveling of underlying molecular, biochemical and physiological mechanisms. We provide here a review of methods used for phenotyping and understanding of abiotic stress (drought/cold) tolerance mechanisms in the context of dynamic challenges faced by plants during their life.

Distinct growth and physiological responses of Arabidopsis thaliana natural accessions to drought stress and their detection using spectral reflectance and thermal imaging

Reduced growth and stomatal closure are the two main responses of plants to drought stress. The extent to which these processes are connected and whether different genotypes prefer one over the other remains unclear. To understand the genotype-specific interconnections of these two processes and evaluate potential utilisation of this knowledge for drought tolerance phenotyping, six natural accessions of Arabidopsis thaliana (L.) Heynh. were exposed to drought stress for 10 days. Projected leaf area of rosette, light-saturated CO2 assimilation rate (Amax), relative water content (RWC), leaf temperature (thermal imaging), and spectral reflectance were measured through the course of induced drought stress. Three types of acclimation were identified: (i) growth not affected but Amax significantly reduced, (ii) both growth and Amax significantly reduced, and (iii) growth significantly reduced but only small decrease in Amax. Within the last type, the smallest decline in RWC was evident. These results show that a substantial reduction in leaf area may cause a decline in transpiration that enables maintenance of both RWC and physiological processes. Both non-invasive thermal imaging and spectral reflectance measurements proved reliable tools for tracking drought-induced changes in Amax and RWC across all accessions tested and thus are effective tools for phenotyping stress tolerance.