Plant morphogenesis relies on the accurate positioning of the partition (cell plate) between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which consists of microtubules, actin filaments, membrane compartments and associated proteins. The phragmoplast forms between daughter nuclei during the transition from anaphase to telophase. As cells are commonly larger than the originally formed phragmoplast, the construction of the cell plate requires phragmoplast expansion. This expansion depends on microtubule polymerization at the phragmoplast forefront (leading zone) and loss at the back (lagging zone). Leading and lagging zones sandwich the ‘transition’ zone. A population of stable microtubules in the transition zone facilitates transport of building materials to the midzone where the cell plate assembly takes place. Whereas microtubules undergo dynamic instability in all zones, the overall balance appears to be shifted towards depolymerization in the lagging zone. Polymerization of microtubules behind the lagging zone has not been reported to date, suggesting that microtubule loss there is irreversible. In this Review, we discuss: (1) the regulation of microtubule dynamics in the phragmoplast zones during expansion; (2) mechanisms of the midzone establishment and initiation of cell plate biogenesis; and (3) signaling in the phragmoplast.

Drought is the leading cause of agricultural yield loss among all abiotic stresses, and the link between water deficit and phloem protein contents is relatively unexplored. Here we collected phloem exudates from Solanum lycopersicum leaves during periods of drought stress and recovery. Our analysis identified 2558 proteins, the most abundant of which were previously localized to the phloem. Independent of drought, enrichment analysis of the total phloem exudate protein profiles from all samples suggests that the protein content of phloem sap is complex, and includes proteins that function in chaperone systems, branched-chain amino acid synthesis, trehalose metabolism, and RNA silencing. We observed 169 proteins whose abundance changed significantly within the phloem sap, either during drought or recovery. Proteins that became significantly more abundant during drought include members of lipid metabolism, chaperone-mediated protein folding, carboxylic acid metabolism, abscisic acid signaling, cytokinin biosynthesis, and amino acid metabolism. Conversely, proteins involved in lipid signaling, sphingolipid metabolism, cell wall organization, carbohydrate metabolism, and a mitogen-activated protein kinase are decreased during drought. Our experiment has achieved an in-depth profiling of phloem sap protein contents during drought stress and recovery that supports previous findings and provides new evidence that multiple biological processes are involved in drought adaptation.

Symbiotic nitrogen fixation (SNF) in the interaction between the soil bacteria Sinorhizobium meliloti and legume plant Medicago sativa is carried out in specialized root organs called nodules. During nodule development, each symbiont must drastically alter their proteins, transcripts and metabolites in order to support nitrogen fixation. Moreover, bacteria within the nodules are under stress, including challenges by plant antimicrobial peptides, low pH, limited oxygen availability, and strongly reducing conditions, all of which challenge proteome integrity. S. meliloti stress adaptation, proteome remodeling and quality control are controlled in part by the large oligomeric protease complexes HslUV and ClpXP1. To improve understanding of the roles of S. meliloti HslUV and ClpXP1 in free-living conditions and in symbiosis with M. sativa, we generated ΔhslU, ΔhslV, ΔhslUV, and ΔclpP1 knockout mutants. Shoot dry weight of M. sativa plants inoculated with each deletion mutant was significantly reduced, suggesting a role in symbiosis. Further, slower free-living growth of ΔhslUV and ΔclpP1 suggest HslUV and ClpP1 were involved in adapting to heat stress, while ΔhslU and ΔclpP1 mutants were sensitive to kanamycin. All deletion mutants produced less exopolysaccharide and succinoglycan, as shown by replicate spot plating and Calcofluor binding. We also generated endogenous C-terminal eGFP fusions to HslU, HslV, ClpX and ClpP1 in S. meliloti. Using anti-eGFP antibodies, native coimmunoprecipitation experiments of proteins from free-living and nodule tissues were performed and analyzed by mass spectrometry. The results suggest HslUV and ClpXP were closely associated with ribosomal and proteome quality control proteins, and identified several novel putative protein-protein interactions. Importance Symbiotic nitrogen fixation (SNF) is the primary means by which biologically available nitrogen enters the biosphere, and is therefore a critical component of the global nitrogen cycle and modern agriculture. SNF is the result of highly coordinated interactions between legume plants and soil bacteria collectively referred to as rhizobia, e.g. Medicago sativa and S. meliloti, respectively. Accomplishing SNF requires significant proteome changes in both organisms to create a microaerobic environment suitable for high-level bacterial nitrogenase activity. Bacterial protease systems, HslUV and ClpXP are important in proteome quality control, metabolic remodeling, and in adapting to stress. This work shows that S. meliloti HslUV and ClpXP are involved in SNF, in exopolysaccharide production, and in free-living stress adaptation.

Symbiotic nitrogen fixation (SNF) between rhizobia and legumes requires metabolic coordination within specialized root organs called nodules. Nodules formed in the symbiosis between S. medicae and barrel medic (M. truncatula) are indeterminate, cylindrical, and contain spatially distinct developmental zones. Bacteria in the infection zone II (ZII), interzone II-III (IZ), and nitrogen fixation zone III (ZIII) represent different stages in the metabolic progression from free-living bacteria into nitrogen fixing bacteroids. To better understand the coordination of plant and bacterial metabolism within the nodule, we used liquid and gas chromatography coupled to tandem mass spectrometry (MS) to observe protein and metabolite profiles representative of ZII, IZ, ZIII, whole-nodule, and primary root. Our MS-based approach confidently identified 361 S. medicae proteins and 888 M. truncatula proteins, as well as 160 metabolites from each tissue. The data are consistent with several organ- and zone-specific protein and metabolite localization patterns characterized previously. We used our comprehensive dataset to demonstrate how multiple branches of primary metabolism are coordinated between symbionts and zones, including central carbon, fatty acid, and amino acid metabolism. For example, M. truncatula glycolysis enzymes accumulate from zone I to zone III within the nodule, while equivalent S. medicae enzymes decrease in abundance. We also show the localization of S. medicae's transition to dicarboxylic acid-dependent carbon metabolism within the IZ. The spatial abundance patterns of S. medicae fatty acid (FA) biosynthesis enzymes indicate an increased demand for FA production in the IZ and ZIII as compared to ZI. These observations provide a resource for those seeking to understand coordinated physiological changes during the development of SNF.

Drought is the largest stress affecting agricultural crops, resulting in substantial reductions in yield. Plant adaptation to water stress is a complex trait involving changes in hormone signaling, physiology, and morphology. Sorghum (Sorghum bicolor (L.) Moench) is a C4 cereal grass; it is an agricultural staple, and it is particularly drought-tolerant. To better understand drought adaptation strategies, we compared the cytosolic- and organelle-enriched protein profiles of leaves from two Sorghum bicolor genotypes, RTx430 and BTx642, with differing preflowering drought tolerances after 8 weeks of growth under water limitation in the field. In agreement with previous findings, we observed significant drought-induced changes in the abundance of multiple heat shock proteins and dehydrins in both genotypes. Interestingly, our data suggest a larger genotype-specific drought response in protein profiles of organelles, while cytosolic responses are largely similar between genotypes. Organelle-enriched proteins whose abundance significantly changed exclusively in the preflowering drought-tolerant genotype RTx430 upon drought stress suggest multiple mechanisms of drought tolerance. These include an RTx430-specific change in proteins associated with ABA metabolism and signal transduction, Rubisco activation, reactive oxygen species scavenging, flowering time regulation, and epicuticular wax production. We discuss the current understanding of these processes in relation to drought tolerance and their potential implications.

In vitro plant propagation systems such as temporary immersion bioreactors (TIBs) are valuable tools that enable production of disease-free plants with improved traits. However, TIB systems can be expensive, difficult to implement, and prone to contamination due to sugar rich propagation media. Using rapidly growing chicory root cultures to expedite design-build-test cycles, we report here an improved, low-cost version of a previously reported Hydrostatically-driven TIB (Hy-TIB) that facilitates economical use of gas mixtures. Bioreactor improvements include decreased material costs, expanded modes of operation, and a horizontal orientation of a plastic film plant growth chambers that increase propagule light exposure. To take advantage of these improvements, we describe here experiments that evaluate the impacts of elevated CO₂ on propagation of cacao (Theobroma cacao) secondary embryos and nodal cultures of yam (Dioscorea spp.) during both phototrophic and photomixotrophic growth. Our experiments show that elevated CO₂ during plant propagation significantly improved both cacao and yam propagule development and eliminated the need for supplemental sugars in tissue culture growth media. Thus, our improved Hy-TIB shows potential as a simple, low-cost, and scalable propagation platform with cost-effective gas composition control and reduced risk of contamination overgrowth. We provide detailed instructions for assembly of this Hy-TIB design and discuss the implications of its adoption in food-insecure regions of the world.