Water scarcity is the primary constraint on crop productivity in arid and semiarid tropical areas suffering from climate alterations; in accordance, agricultural systems have to be optimized. Several concepts and strategies should be considered to improve crop yield and quality, particularly in vulnerable regions where such environmental changes cause a risk of food insecurity. In this work, we review two strategies aiming to increase drought stress tolerance: (i) the use of natural genes that have evolved over time and are preserved in crop wild relatives and landraces for drought tolerance breeding using conventional and molecular methods and (ii) exploiting the reservoir of neglected and underutilized species to identify those that are known to be more drought-tolerant than conventional staple crops while possessing other desired agronomic and nutritive characteristics, as well as introducing them into existing cropping systems to make them more resilient to water deficiency conditions. In the past, the existence of drought tolerance genes in crop wild relatives and landraces was either unknown or difficult to exploit using traditional breeding techniques to secure potential long-term solutions. Today, with the advances in genomics and phenomics, there are a number of new tools available that facilitate the discovery of drought resistance genes in crop wild relatives and landraces and their relatively easy transfer into advanced breeding lines, thus accelerating breeding progress and creating resilient varieties that can withstand prolonged drought periods. Among those tools are marker-assisted selection (MAS), genomic selection (GS), and targeted gene editing (clustered regularly interspaced short palindromic repeat (CRISPR) technology). The integration of these two major strategies, the advances in conventional and molecular breeding for the drought tolerance of conventional staple crops, and the introduction of drought-tolerant neglected and underutilized species into existing production systems has the potential to enhance the resilience of agricultural production under conditions of water scarcity.

Small Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described. The transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal compartments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking. RabA1d is located in early endosomes/TGN and is involved in vesicle trafficking. RabA1d participates in both cell plate formation and root hair oscillatory tip growth. The specific GFP-RabA1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation.

In the present work, we provide an account of structured illumination microscopy (SIM) imaging of fixed and immunolabeled plant probes. We take advantage of SIM, to superresolve intracellular structures at a considerable z-range and circumvent its low temporal resolution capacity during the study of living samples. Further, we validate the protocol for the imaging of fixed transgenic material expressing fluorescent protein-based markers of different subcellular structures. Focus is given on 3D imaging of bulky subcellular structures, such as mitotic and cytokinetic microtubule arrays as well as on the performance of SIM using multichannel imaging and the quantitative correlations that can be deduced. As a proof of concept, we provide a superresolution output on the organization of cortical microtubules in wild-type and mutant Arabidopsis cells, including aberrant preprophase microtubule bands and phragmoplasts in a cytoskeletal mutant devoid of the p60 subunit of the microtubule severing protein KATANIN and refined details of cytoskeletal aberrations in the mitogen activated protein kinase (MAPK) mutant mpk4. We further demonstrate, in a qualitative and quantitative manner, colocalizations between MPK6 and unknown dually phosphorylated and activated MAPK species and we follow the localization of the microtubule associated protein 65-3 (MAP65-3) in telophase and cytokinetic microtubular arrays. 3D SIM is a powerful, versatile and adaptable microscopy method for elucidating spatial relationships between subcellular compartments. Improved methods of sample preparation aiming to the compensation of refractive index mismatches, allow the use of 3D SIM in the documentation of complex plant cell structures, such as microtubule arrays and the elucidation of their interactions with microtubule associated proteins.

Annexin 1 (ANN1) is the most abundant member of the evolutionary conserved multigene protein superfamily of annexins in plants. Generally, annexins participate in diverse cellular processes, such as cell growth, differentiation, vesicle trafficking, and stress responses. The expression of annexins is developmentally regulated, and it is sensitive to the external environment. ANN1 is expressed in almost all Arabidopsis tissues, while the most abundant is in the root, root hairs, and in the hypocotyl epidermal cells. Annexins were also occasionally proposed to associate with cytoskeleton and vesicles, but they were never developmentally localized at the subcellular level in diverse plant tissues and organs. Using advanced light-sheet fluorescence microscopy (LSFM), we followed the developmental and subcellular localization of GFP-tagged ANN1 in post-embryonic Arabidopsis organs. By contrast to conventional microscopy, LSFM allowed long-term imaging of ANN1-GFP in Arabidopsis plants at near-environmental conditions without affecting plant viability. We studied developmental regulation of ANN1-GFP expression and localization in growing Arabidopsis roots: strong accumulation was found in the root cap and epidermal cells (preferentially in elongating trichoblasts), but it was depleted in dividing cells localized in deeper layers of the root meristem. During root hair development, ANN1-GFP accumulated at the tips of emerging and growing root hairs, which was accompanied by decreased abundance in the trichoblasts. In aerial plant parts, ANN1-GFP was localized mainly in the cortical cytoplasm of trichomes and epidermal cells of hypocotyls, cotyledons, true leaves, and their petioles. At the subcellular level, ANN1-GFP was enriched at the plasma membrane (PM) and vesicles of non-dividing cells and in mitotic and cytokinetic microtubular arrays of dividing cells. Additionally, an independent immunolocalization method confirmed ANN1-GFP association with mitotic and cytokinetic microtubules (PPBs and phragmoplasts) in dividing cells of the lateral root cap. Lattice LSFM revealed subcellular accumulation of ANN1-GFP around the nuclear envelope of elongating trichoblasts. Massive relocation and accumulation of ANN1-GFP at the PM and in Hechtian strands and reticulum in plasmolyzed cells suggest a possible osmoprotective role of ANN1-GFP during plasmolysis/deplasmolysis cycle. This study shows complex developmental and subcellular localization patterns of ANN1 in living Arabidopsis plants.