The interplay between pathogens and Atg8 family proteins: thousand-faced interactions.

Autophagy is an intracellular degradation and recycling process that can also remove pathogenic intracellular bacteria and viruses from within cells (referred to as xenophagy) and activate the adaptive immune responses. But autophagy-especially Atg proteins including Atg8 family members-can also have proviral and probacterial effects. In this review, we summarize known interactions of bacterial, parasitic, and viral proteins with Atg8 family proteins and the outcome of these interactions on pathogen replication, autophagy, or mitophagy. We discuss the value of prediction software and the research methodology in the study of pathogen protein-Atg8 family protein interactions, with selected examples of potential LC3-interacting region motif-containing SARS-CoV-2 proteins.

Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles.

Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. In this Brief Report, we use clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. We find that high expression of non-lipidated Atg8b in the male germline is required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility.

E2FA and E2FB transcription factors coordinate cell proliferation with seed maturation.

The E2F transcription factors and the RETINOBLASTOMA-RELATED repressor protein are principal regulators coordinating cell proliferation with differentiation, but their role during seed development is little understood. We show that in fully developed Arabidopsis thaliana embryos, cell number was not affected either in single or double mutants for the activator-type E2FA and E2FB Accordingly, these E2Fs are only partially required for the expression of cell cycle genes. In contrast, the expression of key seed maturation genes LEAFY COTYLEDON 1/2 (LEC1/2), ABSCISIC ACID INSENSITIVE 3, FUSCA 3 and WRINKLED 1 is upregulated in the e2fab double mutant embryo. In accordance, E2FA directly regulates LEC2, and mutation at the consensus E2F-binding site in the LEC2 promoter de-represses its activity during the proliferative stage of seed development. In addition, the major seed storage reserve proteins, 12S globulin and 2S albumin, became prematurely accumulated at the proliferating phase of seed development in the e2fab double mutant. Our findings reveal a repressor function of the activator E2Fs to restrict the seed maturation programme until the cell proliferation phase is completed.

Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion.

The autophagosomal SNARE Syntaxin17 (Syx17) forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS. Here we demonstrate that, unexpectedly, one more SNARE (Ykt6) is also required for autophagosome clearance in Drosophila. We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion. Indeed, Ykt6 localizes to lysosomes and autolysosomes, and forms a SNARE complex with Syx17 and Snap29. Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells. Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms. As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a Syx17-Snap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process.

Proline Accumulation Is Regulated by Transcription Factors Associated with Phosphate Starvation.

Pro accumulation in plants is a well-documented physiological response to osmotic stress caused by drought or salinity. In Arabidopsis (Arabidopsis thaliana), the stress and ABA-induced Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE1 (P5CS1) gene was previously shown to control Pro biosynthesis in such adverse conditions. To identify regulatory factors that control the transcription of P5CS1, Y1H screens were performed with a genomic fragment of P5CS1, containing 1.2-kB promoter and 0.8-kb transcribed regions. The myeloblastosis (MYB)-type transcription factors PHOSPHATE STARVATION RESPONSE1 (PHR1) and PHR1-LIKE1 (PHL1) were identified to bind to P5CS1 regulatory sequences in the first intron, which carries a conserved PHR1-binding site (P1BS) motif. Binding of PHR1 and PHL1 factors to P1BS was confirmed by Y1H, electrophoretic mobility assay and chromatin immunoprecipitation. Phosphate starvation led to gradual increase in Pro content in wild-type Arabidopsis plants as well as transcriptional activation of P5CS1 and PRO DEHYDROGENASE2 genes. Induction of P5CS1 transcription and Pro accumulation during phosphate deficiency was considerably reduced by phr1 and phl1 mutations and was impaired in the ABA-deficient aba2-3 and ABA-insensitive abi4-1 mutants. Growth and viability of phr1phl1 double mutant was significantly reduced in phosphate-depleted medium, while growth was only marginally affected in the aba2-3 mutants, suggesting that ABA is implicated in growth retardation in such nutritional stress. Our results reveal a previously unknown link between Pro metabolism and phosphate nutrition and show that Pro biosynthesis is target of cross talk between ABA signaling and regulation of phosphate homeostasis through PHR1- and PHL1-mediated transcriptional activation of the P5CS1 gene.

Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

Stress tolerance of transgenic barley accumulating the alfalfa aldose reductase in the cytoplasm and the chloroplast.

Barley represents one of the major crops grown worldwide; its genetic transformation provides an important tool for the improvement of crop quality and tolerance to environmental stress factors. Biotic and abiotic stresses produce reactive oxygen species in the plant cells that can directly oxidize the cellular components including lipid membranes; resulting in lipid peroxidation and subsequently the accumulation of reactive carbonyl compounds. In order to protect barley plants from the effects of stress-produced reactive carbonyls, an Agrobacterium-mediated transformation was carried out using the Medicago sativa aldose reductase (MsALR) gene. In certain transgenic lines the produced MsALR enzyme was targeted to the chloroplasts to evaluate its protective effect in these organelles. The dual fluorescent protein-based method was used for the evaluation of tolerance of young seedlings to diverse stresses; our results demonstrated that this technique could be reliably applied for the detection of cellular stress in a variety of conditions. The chlorophyll and carotenoid content measurements also supported the results of the fluorescent protein-based method and the stress-protective effect of the MsALR enzyme. Targeting of MsALR into the chloroplast has also resulted in increased stress tolerance, similarly to the observed effect of the cytosolic MsALR accumulation. The results of the DsRed/GFP fluorescent protein-based method indicated that both the cytosol and chloroplast accumulation of MsALR can increase the abiotic stress tolerance of transgenic barley lines.

Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades.

Mitogen-activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organized into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The Arabidopsis mitogen-activated protein kinase 9 (AtMPK9) is a member of an atypical class of MAPKs. Representatives of this MAPK family have a TDY phosphoacceptor site, a long C-terminal extension and lack the common MAPKK-binding docking motif. In the present paper, we describe multiple in vitro and in vivo data showing that AtMPK9 is activated independently of any upstream MAPKKs but rather is activated through autophosphorylation. We mapped the autophosphorylation sites by MS to the TDY motif and to the C-terminal regulatory extension. We mutated the phosphoacceptor sites on the TDY, which confirmed the requirement for bisphorylation at this site for full kinase activity. Next, we demonstrated that the kinase-inactive mutant form of AtMPK9 is not trans-phosphorylated on the TDY site when mixed with an active AtMPK9, implying that the mechanism of the autocatalytic phosphorylation is intramolecular. Furthermore, we show that in vivo AtMPK9 is activated by salt and is regulated by okadaic acid-sensitive phosphatases. We conclude that the plant AtMPK9 shows similarities to the mammalian atypical MAPKs, such as extracellular-signal-regulated kinase (ERK) 7/8, in terms of an MAPKK-independent activation mechanism.

The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1.

The phosphorylation of plant retinoblastoma-related (RBR) proteins by cyclin-dependent kinases (CDKs) is well documented, but the counteracting phosphatases have not been identified yet. We report here that rice retinoblastoma-related protein-1 (OsRBR1) interacted with the B″ subunit of rice protein phosphatase 2A (OsPP2A B″) and underwent reversible phosphorylation during the cell division cycle. The OsRBR1-OsPP2A B" association required B domain in OsRBR1 and the C-terminal region of OsPP2A B″. We found by immunoprecipitation that OsPP2A B″, OsPP2A catalytic subunit subtype II, PSTAIRE-type CDK and OsRBR1 were in the same protein complex, indicating a physical association between the phosphatase, the kinase and their common substrate. OsPP2A B″ contains three predicted CDK phosphorylation sites: Ser95, Ser102 and Ser119. The in vitro phosphorylation of Ser95 and Ser119 with PSTAIRE-kinases was verified by mass spectrometry. We generated a series of phosphorylation site mutants to mimic the dephosphorylated or phosphorylated states of OsPP2A B″, and confirmed that all of the three predicted sites can be phosphorylated. Yeast two-hybrid experiments suggested that the phosphorylation of OsPP2A B″ promoted the formation of the OsPP2A holoenzyme. A triple phosphorylation mimicking OsPP2A B″ mutant containing holoenzyme showed higher activity in phosphatase assays. Our data collectively show that the phosphatase activity of OsPP2A against OsRBR1 is regulated by the phosphorylation of its B″ regulatory subunit. However, the analysis of the effect of okadaic acid, a phosphatase inhibitor, in rice cell suspension cultures revealed that the dephosphorylation of OsRBR1 was completely inhibited only by high dose (300 nM) of the okadaic acid during the cell cycle progression. Therefore the role of the protein phosphatase 1 should be considered as an additional post translational regulatory component of RBR protein function in higher plants.

Synchronization of Medicago sativa cell suspension culture.

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide the largest amount of biological sample for further analysis. Here we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division and also the application and removal of hydroxyurea blocking agent. A novel method is used for the estimation of cell portion that enters S phase during the assay. The protocol can be used in the case of other species as well.

Cell-cycle control as a target for calcium, hormonal and developmental signals: the role of phosphorylation in the retinoblastoma-centred pathway.

BACKGROUND: During the life cycle of plants, both embryogenic and post-embryogenic growth are essentially based on cell division and cell expansion that are under the control of inherited developmental programmes modified by hormonal and environmental stimuli. Considering either stimulation or inhibition of plant growth, the key role of plant hormones in the modification of cell division activities or in the initiation of differentiation is well supported by experimental data. At the same time there is only limited insight into the molecular events that provide linkage between the regulation of cell-cycle progression and hormonal and developmental control. Studies indicate that there are several alternative ways by which hormonal signalling networks can influence cell division parameters and establish functional links between regulatory pathways of cell-cycle progression and genes and protein complexes involved in organ development. SCOPE: An overview is given here of key components in plant cell division control as acceptors of hormonal and developmental signals during organ formation and growth. Selected examples are presented to highlight the potential role of Ca(2+)-signalling, the complex actions of auxin and cytokinins, regulation by transcription factors and alteration of retinoblastoma-related proteins by phosphorylation. CONCLUSIONS: Auxins and abscisic acid can directly influence expression of cyclin, cyclin-dependent kinase (CDK) genes and activities of CDK complexes. D-type cyclins are primary targets for cytokinins and over-expression of CyclinD3;1 can enhance auxin responses in roots. A set of auxin-activated genes (AXR1-ARGOS-ANT) controls cell number and organ size through modification of CyclinD3;1 gene expression. The SHORT ROOT (SHR) and SCARECROW (SCR) transcriptional factors determine root patterning by activation of the CYCD6;1 gene. Over-expression of the EBP1 gene (plant homologue of the ErbB-3 epidermal growth factor receptor-binding protein) increased biomass by auxin-dependent activation of both D- and B-type cyclins. The direct involvement of auxin-binding protein (ABP1) in the entry into the cell cycle and the regulation of leaf size and morphology is based on the transcriptional control of D-cyclins and retinoblastoma-related protein (RBR) interacting with inhibitory E2FC transcriptional factor. The central role of RBRs in cell-cycle progression is well documented by a variety of experimental approaches. Their function is phosphorylation-dependent and both RBR and phospho-RBR proteins are present in interphase and mitotic phase cells. Immunolocalization studies showed the presence of phospho-RBR protein in spots of interphase nuclei or granules in mitotic prophase cells. The Ca(2+)-dependent phosphorylation events can be accomplished by the calcium-dependent, calmodulin-independent or calmodulin-like domain protein kinases (CDPKs/CPKs) phosphorylating the CDK inhibitor protein (KRP). Dephosphorylation of the phospho-RBR protein by PP2A phosphatase is regulated by a Ca(2+)-binding subunit.

Overproduction of a rice aldo-keto reductase increases oxidative and heat stress tolerance by malondialdehyde and methylglyoxal detoxification.

The accumulation of toxic compounds generated by the interaction between reactive oxygen species and polyunsaturated fatty acids of membrane lipids can significantly damage plant cells. A plethora of enzymes act on these reactive carbonyls, reducing their toxicity. Based on the chromosomal localization and on their homology with other stress-induced aldo-keto reductases (AKRs) we have selected three rice AKR genes. The transcription level of OsAKR1 was greatly induced by abscisic acid and various stress treatments; the other two AKR genes tested were moderately stress-inducible. The OsAKR1 recombinant protein exhibited a high nicotinamide adenine dinucleotide phosphate-dependent catalytic activity to reduce toxic aldehydes including glycolysis-derived methylglyoxal (MG) and lipid peroxidation-originated malondialdehyde (MDA). The function of this enzyme in MG detoxification was demonstrated in vivo in E. coli and in transgenic plants overproducing the OsAKR1 protein. Heterologous synthesis of the OsAKR1 enzyme in transgenic tobacco plants resulted in increased tolerance against oxidative stress generated by methylviologen (MV) and improved resistance to high temperature. In these plants lower levels of MDA were detected both following MV and heat treatment due to the activity of the OsAKR1 enzyme. The transgenic tobaccos also exhibited higher AKR activity and accumulated less MG in their leaves than the wild type plants; both in the presence and absence of heat stress. These results support the positive role of OsAKR1 in abiotic stress-related reactive aldehyde detoxification pathways and its use for improvement of stress tolerance in plants.

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells.

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.

A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine.

BACKGROUND: Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here. RESULTS: Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice. CONCLUSIONS: In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.

Leaf hue measurements offer a fast, high-throughput initial screening of photosynthesis in leaves.

Experiments with tobacco and grapevine leaves having different color due to varying stages of senescence showed that leaf hue is significantly linearly correlated with chlorophyll content up to 80% loss of pigment. Samples from leaves with more pronounced loss of chlorophyll did not fit into this linear relationship, and the hue data set as a whole followed a saturating exponential dependence on chlorophyll content. In leaves with less than 80% chlorophyll loss, the hue parameter was also proportional to the photochemical yield of photosystem (PS) II measured in the light. These results suggest that leaf hue measurements offer a fast, high-throughput initial screening system to precede more specific but more time consuming photosynthesis measurements, with the possibility of applications not only for senescing plants, but also for stress conditions accompanied by chlorophyll loss.

Accumulation of overproduced ferritin in the chloroplast provides protection against photoinhibition induced by low temperature in tobacco plants.

Wild-type tobacco plants (Nicotiana tabacum L. cv. Petit Havanna line SR1) and plants transformed with full-length alfalfa ferritin cDNA with the chloroplast transit peptide under the control of a Rubisco small subunit gene promoter (C3 and C8) were cold-treated at 0 degrees C with continuous light (250 micromol m(-2)s(-1)). These transgenic plants had higher chlorophyll content and higher F(v)/F(m) chlorophyll-a fluorescence induction parameters than wild-type plants after 2 or 3d of cold treatment in C3 and C8 transgenic plants, respectively. Thermoluminescence studies on the high-temperature bands suggest that these plants suffered less oxidative damage in comparison to the wild-type genotype. The present experiments provide evidence that transgenic tobacco lines overexpressing alfalfa ferritin, which is accumulated in the chloroplasts, may show higher tolerance to various stress factors, generating ROS including low temperature-induced photoinhibition.

Imaging fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope.

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence-detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity-difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber-laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture.

3-hydroxy-3-methylglutaryl coenzyme a reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula.

NORK in legumes encodes a receptor-like kinase that is required for Nod factor signaling and root nodule development. Using Medicago truncatula NORK as bait in a yeast two-hybrid assay, we identified 3-hydroxy-3-methylglutaryl CoA reductase 1 (Mt HMGR1) as a NORK interacting partner. HMGR1 belongs to a multigene family in M. truncatula, and different HMGR isoforms are key enzymes in the mevalonate biosynthetic pathway leading to the production of a diverse array of isoprenoid compounds. Testing other HMGR members revealed a specific interaction between NORK and HMGR1. Mutagenesis and deletion analysis showed that this interaction requires the cytosolic active kinase domain of NORK and the cytosolic catalytic domain of HMGR1. NORK homologs from Lotus japonicus and Sesbania rostrata also interacted with Mt HMGR1, but homologous nonsymbiotic kinases of M. truncatula did not. Pharmacological inhibition of HMGR activities decreased nodule number and delayed nodulation, supporting the importance of the mevalonate pathway in symbiotic development. Decreasing HMGR1 expression in M. truncatula transgenic roots by RNA interference led to a dramatic decrease in nodulation, confirming that HMGR1 is essential for nodule development. Recruitment of HMGR1 by NORK could be required for production of specific isoprenoid compounds, such as cytokinins, phytosteroids, or isoprenoid moieties involved in modification of signaling proteins.

Dicot and monocot plants differ in retinoblastoma-related protein subfamilies.

The present study supports the view that the retinoblastoma functions are shared by two distinct retinoblastoma-related (RBR) protein subfamilies in the monocot cereal species, whereas dicot plants have only a single RBR protein. Genes encoding RBR proteins were identified and characterized in alfalfa (Medicago sativa), rice (Oryza sativa), and wheat (Triticum aestivum). The alfalfa MsRBR gene encodes a new member of the dicot RBR proteins (subfamily A). A comparison was made of two rice genes, OsRBR1 (subfamily B) and OsRBR2 (subfamily C), which exhibit differences in exon-intron organization and share only 52% amino acid sequence identity. The plant RBR proteins can be categorized into three distinct subfamilies, in which the similarity between members is greater than the similarity to other RBR proteins from the same species. Comparison of the transcript levels in various tissues revealed that the expression of the OsRBR1 gene was high in embryos or cultured cells and gradually decreased from the basal region to the tip of the leaves. The OsRBR2 gene displayed more transcripts in differentiated tissues, such as leaves and roots. In contrast, the mRNA level of the MsRBR gene did not differ significantly in either mature leaves or cultured cells. The results of yeast two-hybrid pairwise interaction assays demonstrated differences between the rice RBR variants in the interactions with the phosphatase 2A B'' regulatory subunit and an unknown protein. The in silico and functional data presented in this work highlight considerable differences between dicot and monocot species in the retinoblastoma regulatory pathways and permit an improved classification of RBR proteins in higher plants.

Activation of an alfalfa cyclin-dependent kinase inhibitor by calmodulin-like domain protein kinase.

Kip-related proteins (KRPs) play a central role in the regulation of the cell cycle and differentiation through modulation of cyclin-dependent kinase (CDK) functions. We have identified a CDK inhibitor gene from Medicago truncatula (Mt) by a yeast two-hybrid screen. The KRPMt gene was expressed in all plant organs and cultured cells, and its transcripts accumulated after abscisic acid and NaCl treatment. The KRPMt protein exhibits seven conserved sequence domains and a PEST motif that is also detected in various Arabidopsis KRPs. In the yeast two-hybrid test, the KRPMt protein interacted with CDK (Medsa;CDKA;1) and D-type cyclins. However, in the pull-down assays, B-type CDK complexes were also detectable. Recombinant KRPMt differentially inhibited various alfalfa CDK complexes in phosphorylation assays. The immunoprecipitated Medsa;CDKA;1/A;2 complex was strongly inhibited, whereas the mitotic Medsa;CDKB2;1 complex was the most sensitive to inhibition. Function of Medsa;CDKB1;1 complex was not inhibited by the KRPMt protein. The mitotic Medsa;CYCB2 and Medsa;CYCA2;1 complexes responded weakly to this inhibitor protein. Kinase complexes from G2/M cells showed increased sensitivity towards the inhibitor compared with those isolated from G1/S-phase cells. In vitro phosphorylation of Medicago retinoblastoma-related protein was also reduced in the presence of KRPMt. Phosphorylation of this inhibitor protein by the recombinant calmodulin-like domain protein kinase (MsCPK3) resulted in enhanced inhibition of CDK function. The data presented emphasize the selective sensitivity of various cyclin-dependent kinase complexes to this inhibitor protein, and suggest a role for CDK inhibitors and CPKs in cross-talk between Ca2+ signalling and regulation of cell-cycle progression in plants.