Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.

Human population growth and climate change threaten our food and water security. The increasing frequency of extreme drought events will cause major crop yield losses. To mitigate this threat to global food security, we need to rapidly select and/or develop new 'climate-ready' crop varieties that can withstand and flourish under water deficit, enabling the sustained and sustainable production of higher yields to support human life on Earth. In this article, we identify the current targets for crop plant improvement under drought, working from the ground up, with modifications in rooting, shoot, stomatal, and photosynthetic systems, and finally nutrient transport and sink strength. We argue that by using a holistic approach to crop development, prudently incorporating the natural variation available in crop wild relatives and cultivars with cutting-edge tools, such as molecular breeding and transgenics, we may be able to produce high-yielding crops under a range of conditions to meet our needs in a changing world.

A modified protocol was developed to obtain high-quality total RNA from various mature organs, including leaves, seeds, pods and testae, from different cultivars of Phaseolus vulgaris L. grown under optimal conditions or subjected to severe drought; stress conditions leading to the accumulation of numerous secondary metabolites can affect RNA quality. This modified procedure is based on CTAB extraction protocols. Modifications in this protocol prevent oxidation of phenolic complexes, the precipitation of proteins, DNA and degradation of RNA; also, it is effective atremoving secondary metabolites. The RNA obtained following this procedure showed high quality as revealed by a high RNA integrity number and high 260/280nm (>2) ratio, therequirements needed to increase reliability of diverse molecular analyses.

Terminal drought stress affects more than half of the areas planted with common bean (Phaseolus vulgaris), the main food legume globally, generating severe yield losses. Phenotyping water deficit responses and water use are central strategies to develop improved terminal drought resilience. The exploration and exploitation of genetic diversity in breeding programs are gaining importance, with a particular interest in related species with great adaptation to biotic and abiotic factors. This is the case with tepary beans (Phaseolus acutifolius), a bean that evolved and was domesticated in arid conditions and is considered well adapted to drought and heat stress. Under greenhouse conditions, using one genotype of tepary beans (resistant to drought) and two of common beans (one resistant and one susceptible to terminal drought), we evaluated phenotypic differences in traits such as water use efficiency (WUE), transpiration efficiency, rate of photosynthesis, photosynthetic efficiency, stomatal density, stomatal index, stomatal size, and the threshold for transpiration decline under well-watered and terminal drought conditions. Our results indicate two different water use strategies in drought-resistant genotypes: one observed in common bean aimed at conserving soil water by closing stomata early, inhibiting stomatal development, and limiting growth; and the other observed in tepary bean, where prolonged stomatal opening and higher carbon fixation, combined with no changes in stomata distribution, lead to higher biomass accumulation. Strategies that contribute to drought adaptation combined with other traits, such as greater mobilization of photoassimilates to the formation of reproductive structures, confer bean drought resistance and are useful targets in breeding programs.

Common bean (Phaseolus vulgaris L.) is one of the most consumed legumes in the human diet. A major problem for this rainfed crop is the decrease in grain yield caused by prolonged drought periods during the reproductive stage of plant development (terminal drought, TD). Despite the importance of the common bean as a substantial source of proteins, TD remains a prevailing threat to the farming of this staple, with losses reaching more than 80%. Based on the high correlation between the common bean resistance to TD with an efficient photo-assimilate mobilization and biomass accumulation in seeds, we aimed to identify mechanisms implicated in its resistance to this stress. We used two representative Durango race common bean cultivars with contrasting yields under TD, grown under well-watered or TD conditions. By comparative transcriptomic analysis focused on source leaves, pods, and seeds from both cultivars, we provide evidence indicating that under TD the resistant cultivar promotes the buildup of transcripts involved in recycling carbon through photosynthesis, photorespiration, and CO2 concentrating mechanisms in pod walls; while in seeds, the induced transcripts participate in sink strength and respiration. Physiological data support this conclusion, implicating their relevance as key processes in the plant response to TD.