Drought response in wheat is considered a highly complex process, since it is a multigenic
trait; nevertheless, breeding programs are continuously searching for new wheat varieties with
characteristics for drought tolerance. In a previous study, we demonstrated the effectiveness of a
mutant known as RYNO3936 that could survive 14 days without water. In this study, we reveal
another mutant known as BIG8-1 that can endure severe water deficit stress (21 days without water)
with superior drought response characteristics. Phenotypically, the mutant plants had broader leaves,
including a densely packed fibrous root architecture that was not visible in the WT parent plants.
During mild (day 7) drought stress, the mutant could maintain its relative water content, chlorophyll
content, maximum quantum yield of PSII (Fv/Fm) and stomatal conductance, with no phenotypic
symptoms such as wilting or senescence despite a decrease in soil moisture content. It was only
during moderate (day 14) and severe (day 21) water deficit stress that a decline in those variables
was evident. Furthermore, the mutant plants also displayed a unique preservation of metabolic
activity, which was confirmed by assessing the accumulation of free amino acids and increase of
antioxidative enzymes (peroxidases and glutathione S-transferase). Proteome reshuffling was also
observed, allowing slow degradation of essential proteins such as RuBisCO during water deficit stress.
The LC-MS/MS data revealed a high abundance of proteins involved in energy and photosynthesis
under well-watered conditions, particularly Serpin-Z2A and Z2B, SGT1 and Calnexin-like protein.
However, after 21 days of water stress, the mutants expressed ABC transporter permeases and
xylanase inhibitor protein, which are involved in the transport of amino acids and protecting cells,
respectively. This study characterizes a new mutant BIG8-1 with drought-tolerant characteristics
suited for breeding programs.