Attachment of ubiquitin to substrate proteins is catalyzed by the three enzymes E1, E2 (ubiquitin conjugating [UBC]), and E3 (ubiquitin ligase). Forty-one functional proteins with a UBC domain and active-site cysteine are predicted in the Arabidopsis (Arabidopsis thaliana) genome, which includes four that are predicted or shown to function with ubiquitin-like proteins. Only nine were previously characterized biochemically as ubiquitin E2s. We obtained soluble protein for 22 of the 28 uncharacterized UBCs after expression in Escherichia coli and demonstrated that 16 function as ubiquitin E2s. Twelve, plus three previously characterized ubiquitin E2s, were also tested for the ability to catalyze ubiquitination in vitro in the presence of one of 65 really interesting new gene (RING) E3 ligases. UBC22, UBC19-20, and UBC1-6 had variable levels of E3-independent activity. Six UBCs were inactive with all RINGs tested. Closely related UBC8, 10, 11, and 28 were active with the largest number of RING E3s and with all RING types. Expression analysis was performed to determine whether E2s or E3s were expressed in specific organs or under specific environmental conditions. Closely related E2s show unique patterns of expression and most express ubiquitously. Some RING E3s are also ubiquitously expressed; however, others show organspecific expression. Of all the organs tested, RING mRNAs are most abundant in floral organs. This study demonstrates that E2 diversity includes examples with broad and narrow specificity toward RINGs, and that most ubiquitin E2s are broadly expressed with each having a unique spatial and developmental pattern of expression.

Selective modification of proteins by ubiquitination is directed by diverse families of ubiquitin-protein ligases (or E3s). A large collection of E3s use Cullins (CULs) as scaffolds to form multisubunit E3 complexes in which the CUL binds a target recognition subcomplex and the RBX1 docking protein, which delivers the activated ubiquitin moiety. Arabidopsis and rice contain a large collection of CUL isoforms, indicating that multiple CUL-based E3s exist in plants. Here we show that Arabidopsis CUL3a and CUL3b associate with RBX1 and members of the broad complex/tramtrack/bric-a-brac (BTB) protein family to form BTB E3s. Eighty genes encoding BTB domain-containing proteins were identified in the Arabidopsis genome, indicating that a diverse array of BTB E3s is possible. In addition to the BTB domain, the encoded proteins also contain various other interaction motifs that likely serve as target recognition elements. DNA microarray analyses show that BTB genes are expressed widely in the plant and that tissue-specific and isoform-specific patterns exist. Arabidopsis defective in both CUL3a and CUL3b are embryo-lethal, indicating that BTB E3s are essential for plant development.

Calmodulin, a highly conserved protein family that has long been well known as an intracellular calcium sensor, was identified in the culture medium and cell walls of Arabidopsis thaliana suspension-cultured cells by immunoblotting assay. A promotion effect by applying exogenous purified calmodulin and an inhibition effect by the addition of anti-calmodulin anti-serum or calmodulin antagonist to the medium on proliferation of suspension cells were found by monitoring incorporation of [methyl-3H]thymidine into nuclear DNA. Radioligand binding analysis with 35S-labeled calmodulin indicated the presence of specific, reversible, and saturable calmodulin binding sites on the surface of both A. thaliana suspension-cultured cells and its protoplasts; among them at least one is on the surface of Arabidopsis protoplasts, with the Kd～9.2 nM, and two are on the out-surface of Arabidopsis suspension-cultured cells, with Kd values of～47.5 and 830 nM. Chemical crosslinking of 35S-labeled calmodulin to protoplasts revealed 117- and 41-kDa plasma membrane proteins specifically bound to calmodulin, whereas cross-linking with intact suspension-cultured cells verified more calmodulin binding proteins which might be cell wall-associated in addition to membrane-localized. Taking together, our data provide first evidence for the presence of apoplastic calmodulin receptor-like binding proteins on the cell surface of Arabidopsis suspension-cultured cells, which strongly supports our previous idea that apoplastic calmodulin functions as a peptide signal involved in regulation of cell growth and development.

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+- permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with wholecell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, inpollen cells.

The phosphatidylinositol-specific phospholipase C (PIPLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60–65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. colicells show the PIP2-hydrolyzing activity. The RT-PCR analysis,shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PIPLC activity caused by cholera toxin and it also leads to the decrease of [Ca2+]cyt in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by aheterotrimeric G protein and extracellular CaM.