Mitogen-activated protein kinase (MAPK) signaling networks represent important means of signal transduction in plants and other eukaryotes, controlling intracellular signaling by linking perception of environmental or developmental cues to downstream targets. In the Arabidopsis MEKK subfamily, the MKKK19, 20, and 21 form a highly supported clade with the Solanaceous Fertilization-Related Kinases. In Arabidopsis, little is known about this group, except for MKKK20, which is involved in osmotic stress. Using a directed MKKK-MKK yeast two-hybrid (Y2H) screen, MKKK20 was found to interact only with MKK3, while a MKKK20 large-scale Y2H screen retrieved MPK18 as a direct interactant. In vitro phosphorylation assays showed that MKKK20 phosphorylates both MKK3 and MPK18. However, when all three kinases are combined, no synergistic effect is observed on MPK18 phosphorylation, suggesting a direct access to MPK18, consistent with the absence of interaction between MKK3 and MPK18 in protein–protein interaction assays. Since mpk18 mutant plants were previously shown to be defective in microtubule-related functions, phenotypes of mkkk20 single and mkkk20/mpk18 double mutants were investigated to determine if MKKK20 acts upstream of MPK18. This was the case, as mkkk20 root length was shorter than WT in media containing microtubule-disrupting drugs as previously observed for mpk18 plants. Surprisingly, mkk3 plants were also similarly affected, suggesting the presence of two non-complementary pathways involved in Arabidopsis cortical microtubule function, the first including MKKK20, MKK3 and an unknown MPK; the second, a non-canonical MAPK cascade made of MKKK20 and MPK18 that bypasses the need for an MKK intermediate.

SummaryCell-cell communication pervades every aspect of the life of a plant. It is particularly crucial for the development of the gametes and their subtle interaction leading to double fertilization. The ovule is composed of a funiculus, one or two integuments, and a gametophyte surrounded by nucellus tissue. Proper ovule and embryo sac development are critical to reproductive success. To allow fertilization, the correct relative positioning and differentiation of the embryo sac cells are essential. Integument development is also intimately linked with the normal development of the female gametophyte; the sporophyte and gametophyte are not fully independent tissues. Inside the gametophyte, numerous signs of cell-cell communication take place throughout development, including cell fate patterning, fertilization and the early stages of embryogenesis. This review highlights the current evidence of cell-cell communication and signalling elements based on structural and physiological observations as well as the description and characterization of mutants in structurally specific genes. By combining data from different species, models of cell-cell interactions have been built, particularly for the establishment of the germline, for the progression through megagametogenesis and for double fertilization.

These authors contributed equally to this paper. SUMMARYSmall peptides have been shown to regulate numerous aspects of plant development through cell-cell communication. These signaling events are particularly important during reproduction, regulating gamete development and embryogenesis. Rapid alkalinization factor (RALF)-like genes, a large gene family that encodes secreted peptides, have specific or ubiquitous expression patterns. Previously, five RALF-like genes with potential involvement during reproduction were isolated from Solanum chacoense. Here, we show that ScRALF3 is an important peptide regulator of female gametophyte development. Its expression, which is auxin-inducible, is strictly regulated before and after fertilization. Down-regulation of ScRALF3 expression by RNA interference leads to the production of smaller fruits that produce fewer seeds, due to improper development of the embryo sacs. Defects include loss of embryo sac nuclei polarization, as well as an increase in asynchronous division, accounting for cellular dysfunctions and premature embryo sac development arrest during megagametogenesis. ScRALF3 is expressed in the sporophytic tissue surrounding the embryo sac, the integument and the nucellus, as revealed by in situ hybridization and GUS staining. As expected for a secreted peptide, fluorescence from an ScRALF3-GFP fusion construct is detected throughout the secretory pathway. Therefore, the ScRALF3 secreted peptide may be directly involved in the regulation of multiple aspects of cell-cell communication between the female gametophyte and its surrounding sporophytic tissue during ovule development.

There are increasing concerns about trace metal levels such as copper (Cu) in industrial sites and the broader environment. Different studies have highlighted the role of mycorrhizal associations in plant tolerance to trace metals, modulating some of the plant metabolic and physiological responses. In this study, we investigated the role of the symbiotic association betweenRhizophagus irregularisandSalix purpureaL. in modulating plant responses under Cu stress. We measured Cu accumulation, oxidative stress-related, photosynthetic-related and hydraulic traits, for non-inoculated (non-arbuscular mycorrhizal fungi) and inoculated saplings exposed to different Cu concentrations. We found thatS. purpureais a suitable option for phytoremediation of Cu, acting as a phytostabilizer of this trace metal in its root system. We observed that the symbiotic association modulates a broad spectrum of metabolic and physiological responses inS. purpureaunder Cu conditions, including (i) a reduction in gas exchange associated with chlorophyll content changes and (ii) the sequestration of Cu into the cell walls, modifying vessels anatomy and impacting leaf specific conductivity (KL) and root hydraulic conductance (LP). UpholdingKLandLPunder Cu stress might be related to a dynamic Aquaporin gene regulation ofPIP1;2along with an up-regulation ofTIP2;2in the roots of inoculatedS. purpurea.

Signalling events through small peptides are essential in multiple aspects of plant reproduction. The ScRALF3 Solanum chacoense Rapid Alkalinization Factor (RALF) peptide was previously shown to regulate multiple aspects of cell–cell communication between the surrounding sporophytic tissue and the female gametophyte during ovule development. We analysed the global expression pattern of ScRALF3 with GUS reporter gene under control of the ScRALF3 promoter and validated it with in situ hybridisation. To better understand the role of ScRALF3 we used three different RNA interference (RNAi) lines that reduced the expression of ScRALF3 during pollen development. Both expression methods showed the presence of ScRALF3 in different tissues, including stigma, style, vascular tissues and during stamen development. Down‐regulation of ScRALF3 expression through RNAi showed drastic defects in early stages of pollen development, mainly on the first mitosis. These results suggest that the ScRALF3 secreted peptide regulates the transition from sporogenesis to gametogenesis in both male and female gametophytes.