Calcium ions are used as ubiquitous, key second messengers in cells across eukaryotic taxa. In plants, calcium signal transduction is involved in a wide range of cellular processes from abiotic and biotic stress responses to development and growth. Calcium signals are detected by calcium sensor proteins, of which calmodulin (CaM), is the most evolutionarily conserved and well-studied. These sensors regulate downstream targets to propagate the information in signaling pathways. Plants possess a large family of calcium sensors related to CaM, termed CaM-like (CMLs), that are not found in animals and remain largely unstudied at the structural and functional level. Here, we investigated the biochemical properties and gene promoter activity of two closely related members of the Arabidopsis CML family, CML15 and CML16. Biochemical characterization of recombinant CML15 and CML16 indicated that they possess properties consistent with their predicted roles as calcium sensors. In the absence of calcium, CML15 and CML16 display greater intrinsic hydrophobicity than CaM. Both CMLs displayed calcium-dependent and magnesium-independent conformational changes that expose hydrophobic residues, but the degree of hydrophobic exposure was markedly less than that observed for CaM. Isothermal titration calorimetry indicated two and three calcium-binding sites for CML15 and CML16, respectively, with affinities expected to be within a physiological range. Both CML15 and CML16 bound calcium with high affinity in the presence of excess magnesium. Promoter-reporter analysis demonstrated that the CML16 promoter is active across a range of Arabidopsis tissues and developmental stages, whereas the CML15 promoter activity is very restricted and was observed only in floral tissues, specifically anthers and pollen. Collectively, our data indicate that these CMLs behave biochemically like calcium sensors but with properties distinct from CaM and likely have non-overlapping roles in floral development. We discuss our findings in the broader context of calcium sensors and signaling in Arabidopsis.

Pisum sativum L. (pea) root nodule cells undergo many cellular changes in response to infection by Rhizobium leguminosarum bv. viciae. These include cell growth, organelle reorganization, and changes relating to the increase in the number of bacteria within the cell. The objective of this study was to characterize microtubule organization during nodule cell development. The organization of microtubules was examined in developing pea root nodules using fluorescence and electron microscopy techniques. Immunolabelling of microtubules in meristematic cells showed diffuse fluorescence in the cell cortex and adjacent to the nuclear envelope. Recently infected cells contained randomly oriented cortical microtubules and cytoplasmic microtubules that were fragmented with diffuse fluorescence. Infected cells contained an extensive network of long, randomly arranged cortical microtubules with some parallel bundles. Cytoplasmic microtubules in single optical sections of infected cells appeared as short undulating filaments; however, overlapping images from a Z-series of an infected cell showed that the microtubules are long and wavy, and generally radiate inward from the cell cortex.Key words: nodule, microtubules, Rhizobium, pea, symbiosis.

Summary. Pea (Pisum sativum) root nodule cells infected by the diazotroph Rhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and highpressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.

Various microorganisms that form symbiotic associations with plant roots alter the cytoskeleton of host cells. The objective of this study was to determine the organization of actin microfilaments in developing Pisum sativum L. (pea) root nodule cells at various stages after infection by Rhizobium leguminosarum bv. viciae. Fluorescently labelled microfilaments in uninfected pea root nodule cells occur in association with the nucleus, along cytoplasmic strands, and as long microfilament bundles randomly organized in the cortex of the cell. These actin arrays are also present in recently infected cells that have been invaded by an infection thread and contain a small number of bacteroids. In addition, the recently infected cells contain diffuse cytoplasmic actin, long actin microfilament bundles near the vacuole, and a nuclear-associated network of microfilament bundles. In older infected cells, the predominant array is a network of cytoplasmic microfilaments that are wavy and extend in multiple directions within the cell; the network is equally abundant in all regions of the cytoplasm and may interact with the bacteroids and organelles. Thus, actin microfilaments reorganize during the pea root nodule infection process to form distinct arrays whose organization depends on the stage of infection.Key words: nodule, actin microfilaments, Rhizobium, pea, symbiosis.