Flawed evaluation of drought resistance is still hampering transgenic research

Hsieh et al. from the Institute of BioAgricultural Sciences, Academia Sinica, Nankang, Taipei, Taiwan published a paper in ‘Plant Physiology’ journal, which claimed to prove that a transgenic tomato carrying the CBF1 factor was more resistant to water stress as compared with the wild type plant (see abstract below*). The main proof was in that the transgenic plants had delayed wilting, delayed stomatal closure and had higher % water content in leaves as compared with the wild type, with some associated effects such as a respective difference in chlorophyll fluorescence etc.’.

The most outstanding result of the reported genetic transformation was that the CBF1 transgenic (C) plants expressed what the authors defined as a “dwarf phenotype”, being much smaller than the wild type (WT) (Fig.1).

Small plants use less water than larger plants simply as a function of the respective difference in their leaf area. When grown in pots of a given volume, such as in this study and when irrigation is stopped to initiate water stress, larger (WT) plants will express wilting symptoms and stomatal closure earlier than the smaller (C) plants - by the token of their respectively different size and rate of water use. The reader of this paper is therefore allowed to assume that the difference in plant size between WT and C plants was the main reason for the delayed wilting of C plants in this experiment and it masked any effect that CBF1 may or may not had on plant response to water stress. Furthermore, smaller (“dwarf”) transgenic plants may be a result (as commonly observed) of degeneration and stunting associated with the transgenic event rather than from a direct expression of the gene in question. There is no indication here to discount the possibility that CBF1 transgenic plants were simply degenerated and as such required little water when grown in a pot.

Taken at face value the actual core result may indicate only that CBF1 reduces water use in potted plants simply because it conferred a small phenotype. Real proof of the effect of CBF1 on physiological or biochemical factors ascribing drought tolerance (as claimed by Hsieh et al.) must be explored in isolation of its effect on plant morphology and the possible effect of the transgenic event towards plant degeneration.

Secondly, the authors chose to use “% water content” in leaves (on a dry matter basis) as the measure of plant water status. This measure is unknown in plant physiology. It cannot be used to assess plant water status simply because differences in leaf assimilation and structure between genotypes can affect % water content and that has nothing to do with plant water relations. Evidently the authors (and the reviewers of this paper at ‘Plant Physiology’) were not aware of the standard (textbook) tests for plant water status such as ‘relative water content’ or ‘leaf water potential’.

(*) Tsai-Hung Hsieh, Jent-turn Lee, Yee-yung Charng, and Ming-Tsair Chan (2002). Tomato Plants Ectopically Expressing Arabidopsis CBF1 Show Enhanced Resistance to Water Deficit Stress. Plant Physiol. 130:618-626.

ABSTRACT

A DNA cassette containing an Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) cDNA and a nos terminator, driven by a cauliflower mosaic virus 35S promoter, was transformed into the tomato (Lycopersicon esculentum) genome. These transgenic tomato plants were more resistant to water deficit stress than the wild-type plants. The transgenic plants exhibited growth retardation by showing dwarf phenotype, and the fruit and seed numbers and fresh weight of the transgenic tomato plants were apparently less than those of the wild-type plants. Exogenous gibberellic acid treatment reversed the growth retardation and enhanced growth of transgenic tomato plants, but did not affect the level of water deficit resistance. The stomata of the transgenic CBF1 tomato plants closed more rapidly than the wild type after water deficit treatment with or without gibberellic acid pretreatment. The transgenic tomato plants contained higher levels of Pro than those of the wild-type plants under normal or water deficit conditions. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in transgenic tomato plants and the CAT1 (CATALASE1) was characterized. Catalase activity increased, and hydrogen peroxide concentration decreased in transgenic tomato plants compared with the wild-type plants with or without water deficit stress. These results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants.