Drought is one of the major stress factors affecting the growth and development of plants. In this context, drought-related losses of crop plant productivity impede sustainable agriculture all over the world. In general, plants respond to water deficits by multiple physiological and metabolic adaptations at the molecular, cellular, and organism levels. To understand the underlying mechanisms of drought tolerance, adequate stress models and arrays of reliable stress markers are required. Therefore, in this review we comprehensively address currently available models of drought stress, based on culturing plants in soil, hydroponically, or in agar culture, and critically discuss advantages and limitations of each design. We also address the methodology of drought stress characterization and discuss it in the context of real experimental approaches. Further, we highlight the trends of methodological developments in drought stress research, i.e., complementing conventional tests with quantification of phytohormones and reactive oxygen species (ROS), measuring antioxidant enzyme activities, and comprehensively profiling transcriptome, proteome, and metabolome.

A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation.

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

Phenotypic characterization of pea symbiotic mutants has provided a detailed description of the symbiosis with Rhizobium leguminosarum bv. viciae strains. We show here that two allelic non-nodulating pea mutants, RisNod4 and K24, are affected in the PsSym37 gene, encoding a LysM receptor kinase similar to Lotus japonicus NFR1 and Medicago truncatula LYK3. Phenotypic analysis of RisNod4 and K24 suggests a role for the SYM37 in regulation of infection-thread initiation and nodule development from cortical-cell division foci. We show that RisNod4 plants carrying an L to F substitution in the LysM1 domain display a restrictive symbiotic phenotype comparable to the PsSym2^A lines that distinguish ‘European’ and ‘Middle East’ Rhizobium leguminosarum bv. viciae strains. RisNod4 mutants develop nodules only in the presence of a ‘Middle East’ Rhizobium strain producing O-acetylated Nod factors indicating the SYM37 involvement in Nod-factor recognition. Along with the PsSym37, a homologous LysM receptor kinase gene, PsK1, was isolated and characterized. We show that PsK1 and PsSym37 are genetically linked to each other and to the PsSym2 locus. Allelic complementation analyses and sequencing of the extracellular regions of PsSym37 and PsK1 in several ‘European’ and ‘Afghan’ pea cultivars point towards PsK1 as possible candidate for the elusive PsSym2 gene.

KNOX transcription factors (TFs) regulate different aspects of plant development essentially through their effects on phytohormone metabolism. In particular, KNOX TF SHOOTMERISTEMLESS activates the cytokinin biosynthesis ISOPENTENYL TRANSFERASE (IPT) genes in the shoot apical meristem. However, the role of KNOX TFs in symbiotic nodule development and their possible effects on phytohormone metabolism during nodulation have not been studied to date. Cytokinin is a well-known regulator of nodule development, playing the key role in the regulation of cell division during nodule primordium formation. Recently, the activation of IPT genes was shown to take place during nodulation. Therefore, it was hypothesized that KNOX TFs may regulate nodule development and activate cytokinin biosynthesis upon nodulation. This study analysed the expression of different KNOX genes in Medicago truncatula Gaertn. and Pisum sativum L. Among them, the KNOX3 gene was upregulated in response to rhizobial inoculation in both species. pKNOX3::GUS activity was observed in developing nodule primordium. KNOX3 ectopic expression caused the formation of nodule-like structures on transgenic root without bacterial inoculation, a phenotype similar to one described previously for legumes with constitutive activation of the cytokinin receptor. Furthermore, in transgenic roots with MtKNOX3 knockdown, downregulation of A-type cytokinin response genes was found, as well as the MtIPT3 and LONELYGUY2 (MtLOG2) gene being involved in cytokinin activation. Taken together, these findings suggest that KNOX3 gene is involved in symbiotic nodule development and may regulate cytokinin biosynthesis/activation upon nodule development in legume plants.

Drought is one of the major stress factors affecting growth and development of plants. In this context, drought-related losses of crop plant productivity impede sustainable agriculture all over the world. In general, plants responses to water deficit by multiple physiological and metabolic adaptations at the molecular, cellular and organism levels. To understand the underlying mechanisms of drought tolerance, adequate stress models and arrays of reliable stress markers are required. Therefore, in this review we comprehensively address currently available models of drought stress, based on culturing plants in soil, hydroponic or agar culture. These experimental setups give access to different aspects of plant response to drought, like decrease of tissue water potential, reduction of stomata conductance and photosynthesis efficiency, accumulation of low-molecular weight solutes (metabolic adjustment) and drought protective proteins. Till now, this pattern of markers was successfully extended to the methods of enzyme chemistry, molecular biology and omics techniques. Thus, conventional tests can be efficiently complemented by determination of phytohormone and reactive oxygen species (ROS) contents, activities of antioxidant enzymes, as well as comprehensive profiling of transcriptome, proteome and metabolome.