Transcriptome and anatomical studies reveal alterations in leaf thickness under long-term drought stress in tobacco

Drought is one of the foremost environmental factors that limit the growth of plants. Leaf thickness (LT) is an important quantitative trait in plant physiology. The experiment was carried out in a growth room and the plants were divided into two groups such as well-watered and drought-stressed. This work investigated leaf growth in terms of leaf surface growth and expansion rate, leaf stomata traits, LT, anticlinal growth, and leaf cell layers. The results showed that the leaf area and leaf surface expansion rate were decreased by drought stress (DS). Similarly, LT, anticlinal expansion rate, palisade and spongy tissue thickness, and their related expansion rates were also decreased at different days' time points (DTP) of DS. However, a steady increase was observed in the aforementioned parameters after 12 DTP of DS. The stomatal density increased while stomata size decreased at 3 DTP and 12 DTP (low leaf water potential and relative leaf water content at these time points) and vice versa at 24 DTP compared with the well-watered plants indicating adaptations in these traits in response to DS, and thus the leaf water status played a role in the regulation of leaf stomata traits. The cell length decreased in the upper epidermis, palisade and spongy tissues by DS up to 12 DTP led to lower LT while an increase was observed after 12 DTP that resulted in higher LT. The increase in the LT was supported by the upregulation of starch and sucrose metabolism, glycerolipid metabolism, protein processing in endoplasmic reticulum pathways at 18 DTP along with the differentially expressed genes induced that were related to cell wall remodeling (cellulose, expansin, xyloglucans) and cell expansion (auxin response factors and aquaporin). The results explain the response of leaf thickness to drought stress and show alterations in LT and leaf stomatal traits. This study might serve as a valuable source of gene information for functional studies and provide a theoretical basis to understand leaf growth in terms of leaf anatomy and leaf stomatal traits under drought stress.