Non-vernalization requirement for flowering in Brassica rapa conferred by a dominant allele of FLOWERING LOCUS T.

KEY MESSAGE: We identified and characterized a dominant FT allele for flowering without vernalization in Brassica rapa, while demonstrating its potential for deployment in breeding to accelerate flowering in various Brassicaceae crops. Controlling the timing of flowering is key to improving yield and quality of several agricultural crops including the Brassicas. Many Brassicaceae crops possess a conserved flowering mechanism in which FLOWERING LOCUS C (FLC) represses the transcription of flowering activators such as FLOWERING LOCUS T (FT) during vernalization. Here, we employed genetic analysis based on next-generation sequencing to identify a dominant FT allele, BraA.FT.2-C, for flowering in the absence of vernalization in the Brassica rapa cultivar 'CHOY SUM EX CHINA 3'. BraA.FT.2-C harbors two large insertions upstream of its coding region and is expressed without vernalization, despite FLC expression. We show that BraA.FT.2-C offers an opportunity to introduce flowering without vernalization requirement into winter-type brassica crops, including B. napus, which have many functional FLC paralogs. Furthermore, we demonstrated the feasibility of using B. rapa harboring BraA.FT.2-C as rootstock for grafting to induce flowering in radish (Raphanus sativus), which requires vernalization for flowering. We believe that the ability of BraA.FT.2-C to overcome repression by FLC can have significant applications in brassica crops breeding to increase yields by accelerating or delaying flowering.

A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.

Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss Physcomitrium (Physcomitrella) patens, the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in Arabidopsis thaliana control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.

Cryptogein-induced cell cycle arrest at G2 phase is associated with inhibition of cyclin-dependent kinases, suppression of expression of cell cycle-related genes and protein degradation in synchronized tobacco BY-2 cells.

Induction of defense responses by pathogens or elicitors is often accompanied by growth inhibition in planta, but its molecular mechanisms are poorly understood. In this report, we characterized the molecular events that occur during cryptogein-induced cell cycle arrest at G(2) phase in synchronously cultured tobacco Bright Yellow-2 (BY-2) cells. Concomitant with the proteinaceous elicitor-induced G(2) arrest, we observed inhibition of the histone H1 kinase activity of cyclin-dependent kinases (CDKs), which correlated with a decrease in mRNA and protein levels of CDKB1. In contrast, the amount of CDKA was almost unaffected by cryptogein even at M phase. Cryptogein rapidly inhibited the expression not only of positive, e.g. A- and B-type cyclins and NtCAK, but also of negative cell cycle regulators such as WEE1, suggesting that cryptogein affects multiple targets to inactivate CDKA to induce G(2) arrest by mechanisms distinct from known checkpoint regulation. Moreover, we show that CDKB1 and cyclin proteins are also rapidly degraded by cryptogein and that the proteasome-dependent protein degradation has a crucial role in the control of cryptogein-induced hypersensitive cell death.

Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit translation is regulated in a small subunit-independent manner in the expanded leaves of tobacco.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of small subunits (SSs) encoded by rbcS on the nuclear genome and large subunits (LSs) encoded by rbcL on the chloroplast genome, and it is localized in the chloroplast stroma. Constitutive knockdown of the rbcS gene reportedly causes a reduction in LS quantity and the level of translation in tobacco and the unicellular green alga Chlamydomonas. Constitutively knockdown of the rbcS gene also causes a reduction in photosynthesis, which influences the expression of photosynthetic genes, including the rbcL gene. Here, to investigate the influence of the knockdown of the rbcS gene on the expression of the rbcL gene under normal photosynthetic conditions, we generated transgenic tobacco plants in which the amount of endogenous rbcS mRNA can be reduced by inducible expression of antisense rbcS mRNA with dexamethasone (DEX) treatment at later stages of growth. In already expanded leaves, after DEX treatment, the level of photosynthesis, RuBisCO quantity and the chloroplast ultrastructure were normal, but the amount of rbcS mRNA was reduced. An in vivo pulse labeling experiment and polysome analysis showed that LSs were translated at the same rate as in wild-type leaves. On the other hand, in newly emerging leaves, the rbcS mRNA quantity, the level of photosynthesis and the quantity of RuBisCO were reduced, and chloroplasts failed to develop. In these leaves, the level of LS translation was inhibited, as previously described. These results suggest that LS translation is regulated in an SS-independent manner in expanded leaves under normal photosynthetic conditions.

Auxin is required for the assembly of A-type cyclin-dependent kinase complexes in tobacco cell suspension culture.

Although activation of A-type cyclin-dependent kinase (CDKA) is required for plant cell division, little is known about how CDKA is activated before commitment to cell division. Here, we show that auxin is required for the formation of active CDKA-associated complexes, promoting assembly of the complex in tobacco suspension culture Bright Yellow-2 (BY-2) cells. Protein gel blot analysis revealed that CDKA levels increased greatly after stationary-phase BY-2 cells were subcultured into fresh medium to re-enter the cell cycle. However, these increasing levels subsided when cells were subcultured into auxin-deprived medium, and a subtle increase was observed after subculturing into sucrose-deprived medium. Additionally, p13(suc1)-associated kinase activity did not increase significantly after subculturing into either auxin- or sucrose-deprived medium, but increased strongly after subculturing into medium containing both auxin and sucrose. Using gel filtration, we found that p13(suc1)-associated kinase activity against tobacco retinoblastoma-related protein was maximal in fractions corresponding to the molecular mass of the cyclin/CDKA complex. Interestingly, this peak distribution of high molecular-mass fractions of CDKA disappeared after cells were subcultured into auxin-deprived medium. These findings suggest an important role for auxin in the assembly of active CDKA-associated complexes.

Arabidopsis KRPs have distinct inhibitory activity toward cyclin D2-associated kinases, including plant-specific B-type cyclin-dependent kinase.

Arabidopsis contains seven Kip-related protein (KRP) genes encoding CDK (cyclin-dependent kinase) inhibitors (CKIs), which shares a restricted similarity with mammalian p27Kip1. Here, we analyze the characteristics of the KRPs. Although KRP1-KRP7 interact with active cyclin D2 (CYCD2)/CDKA and CYCD2/CDKB complexes to a similar extent, they inhibit kinase activity to a different extent. Our results suggest that inhibitory activity is related to the binding ability between KRP proteins and cyclin/CDK complexes, but secondary and tertiary structure may be also involved. These data provide the first evidence that KRPs inhibit kinase activity associated with plant-specific CDKB.

5' Untranslated region of the HSP18.2 gene contributes to efficient translation in plant cells.

Maximizing the amount of protein translated per unit mRNA is an important goal in establishing expression systems. The 5' untranslated region (5'-UTR) of mRNA is known to play an important role in determining the rate of translation. The full length 5'-UTR from the Arabidopsis thaliana heat shock protein (HSP) gene,HSPI8.2 gene, was inserted into the cloning site between the cauliflower mosaic virus 35S RNA promoter and beta-glucuronidase (GUS) gene in the pBI221 plasmid. When this construct was transfected into Nicotiana tabacum (tobacco) BY-2 protoplasts, the level of protein was about 10-fold higher than that of unmodified pB1221. The accumulation of each transcript was the same level. We also demonstrated that the 5'-UTR of the HSP18.2 gene enhances the rate of translation in stable transgenic BY-2 clones and Arabidopsis T87 protoplasts. The 5'-UTRs of the other Arabidopsis HSP genes -HSP17.4, HSP81-1,HSP81-2, andHSP81-3 - also conferred efficient translation. These 5'-UTRs ofHSP genes may be of use in increasing the expression of foreign proteins. In combination with a strong promoter, it can be used in the development of efficient protein production systems.

Measurement of plant cyclin-dependent kinase activity using immunoprecipitation-coupled and affinity purification-based kinase assays and the baculovirus expression system.

Orderly progression of the eukaryotic cell cycle is governed by a coordinated response to intrinsic and extracellular cues through activation of cyclin-dependent kinases (CDKs). It is therefore important to verify the kinase activity of distinct types of CDKs during the cell cycle. The immunoprecipitation-coupled kinase assay is a useful procedure to evaluate CDK activity in vivo. Although a specific antibody is usually required for immunoprecipitation, transgenic plant cells expressing tag- or marker protein-fused CDKs are also suitable for this purpose. In addition, the baculovirus expression system is a valuable tool for analyzing CDK activity in vitro, because activation of CDKs is regulated by posttranscriptional modification systems that are active in the insect host cells.

Both negative and positive G1 cell cycle regulators undergo proteasome-dependent degradation during sucrose starvation in Arabidopsis.

The proteasome pathway regulates many aspects of biological processes in plants, such as plant hormone signaling, light responses, the circadian clock and regulation of cell division. Key cell-cycle regulatory proteins including B-type cyclins, Cdc6, cyclin-dependent kinase inhibitors and E2Fc undergo proteasome-dependent degradation. We used the proteasome inhibitor MG132 to show that proteolysis of Arabidopsis RETINOBLASTOMA-RELATED 1 (AtRBR1) and three E2Fs is mediated by the proteasome pathway during sucrose starvation in Arabidopsis suspension MM2d cells. We found previously that estrogen-inducible RNAi-mediated downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Degradation of not only negative (AtRBR1 and E2Fc) but also positive (E2Fa and E2Fb) cell cycle regulators after sucrose starvation may be required for arrest in G1 phase, when cells integrate a variety of nutritional, hormonal and developmental signals to decide whether or not to commit to entry into the cell cycle.

Arabidopsis G1 cell cycle proteins undergo proteasome-dependent degradation during sucrose starvation.

Although sucrose availability is crucial for commitment to plant cell division during G1 phase, it has remained uncertain how protein levels of core cell cycle genes are regulated. We found that Arabidopsis retinoblastoma-related protein1 (AtRBR1) and three E2F proteins were degraded under limited sucrose conditions, while protein abundance increased in response to treatment with the proteasome inhibitor MG132. We conclude that Arabidopsis key cell cycle proteins are degraded in a proteasome-dependent manner during sucrose starvation in Arabidopsis suspension MM2d cells.

Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 is involved in G1 phase cell cycle arrest caused by sucrose starvation.

Although sucrose availability is crucial for commitment to plant cell division during G1 phase by controlling the expression of D-type cyclins, it has remained unclear how these factors mediate entry into the cell cycle. Here we show that Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 (AtRBR1) is involved in G1-phase cell cycle arrest caused by sucrose starvation. We generated estrogen-inducible AtRBR1 RNA interference (RNAi) Arabidopsis suspension MM2d cells, and found that downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Synchronization experiments confirmed that downregulation of AtRBR1 leads to a prolonged G2 phase and delayed activation of G2/M marker genes. Downregulation of AtRBR1 also stimulated the activation of E2F-regulated genes when these genes were repressed in the uninduced cells under the limited sucrose conditions. We conclude that AtRBR1 is a key effector for the ability of sucrose to modulate progression from G1 phase.

Phosphorylation of threonine 161 in plant cyclin-dependent kinase A is required for cell division by activation of its associated kinase.

Although A-type cyclin-dependent kinase A (CDKA) is required for plant cell division, our understanding of how CDKA is activated before the onset of commitment to cell division is limited. Here we show that phosphorylation of threonine 161 (T161) in plant CDKA is required for activation of its associated kinase. Western blot analysis revealed that phosphorylation of CDKA T161 increased greatly, in parallel with activation of p13(suc1)-associated kinase activity, when stationary-phase tobacco BY-2 cells were subcultured into fresh medium. Although induced over-expression of a dominant-negative CDKA mutant (D146N) fused with green fluorescent protein (GFP) in BY-2 cells resulted in elongated cells after cell division was arrested, over-expression of this CDKA mutant with a non-phosphorylatable alanine in place of T161 (T161A) had no effect on cellular growth. However, immunoprecipitates of both GFP-fused CDKAs exhibited virtually no histone H1 kinase activity, suggesting that both mutants formed kinase-inactive complexes. In a baculovirus expression system, the recombinant CDKA(T161A)/cyclin D complex possessed no detectable kinase activity, indicating that phosphorylation of T161 is required for CDKA activation. To further elucidate the role of T161 phosphorylation, we used a loss-of-function mutation in the CDKA;1 gene, which encodes the only Arabidopsis CDKA. This mutant displays male gametophyte lethality, and produces bicellular pollen grains instead of the tricellular grains produced in wild-type plants. Introduction of CDKA;1(T161E)-GFP, which mimics phosphorylated T161, resulted in successful complementation of the cdka-1 mutation, whereas no recovery was observed when CDKA;1(T161A)-GFP was introduced. Thus, phosphorylation of T161 in Arabidopsis CDKA;1 is essential for cell division during male gametogenesis.

Arabidopsis CDKA;1, a cdc2 homologue, controls proliferation of generative cells in male gametogenesis.

The protein kinase cdc2 is conserved throughout eukaryotes and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. Here we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in lethality of the male gametophyte. Heterozygous plants produced mature siliques containing about 50% aborted seeds, and segregation distortion was observed in paternal inheritance. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The larger vegetative cell does not divide, but the smaller generative cell undergoes mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. The mutant sperm-like cell is fertile, and preferentially fuses with the egg cell to initiate embryogenesis. As the central cell nucleus remains unfertilized, however, double fertilization does not occur. In heterozygous plants, the embryo is arrested at the globular stage, most likely because of loss of endosperm development, whereas it is arrested at the one- or two-cell stage in presumptive homozygous plants. Thus, CDKA;1 is essential for cell division of the generative cell in male gametogenesis.

Cell cycle regulated D3-type cyclins form active complexes with plant-specific B-type cyclin-dependent kinase in vitro.

Tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cells are the most highly synchronizable plant cell culture, and previously we used them to analyze cell cycle regulation of cyclin-dependent kinases (CDKs) containing the cyclin binding motifs PSTAIRE (CDKA) and PPTA/TLRE (CDKB). Here we describe the analysis of tobacco CycD3 cyclins whose transcripts predominantly accumulate during G2 to M phase, which represents a unique feature of this type of cyclin D in plants. Although protein levels of CycD3s fluctuate with different patterns during the cell cycle, kinase assays revealed that the CycD3-associated kinases phosphorylate histone H1 and the tobacco retinoblastoma related protein (NtRBR1) with two peaks at the G1/S and G2/M boundaries. In vitro pull-down assays revealed that cell cycle-regulated CycD3s bind to CDKA, but more weakly than does CycD3;3, and that they also bind to CDKB and the CDK inhibitor NtKIS1a. Mutations in the cyclin box of the CycD3s showed that two amino acids are required for binding with CDKA and NtKIS1a, but no diminished interaction was observed with CDKB. A reconstituted kinase assay was adapted for use with bacterially produced GST-CycD3s, and kinase activity could be activated by incubation of extracts from exponentially growing BY-2 cells. Such activated complexes contained CDKA and CDKB, and the reconstituted GST-CycD3 mutants, retaining binding ability to CDKB, showed kinase activity, suggesting that these cell cycle-regulated CycD3s form active complexes with both A- and B-type CDKs in vitro.

Expression of randomly integrated single complete copy transgenes does not vary in Arabidopsis thaliana.

The high variability of transgene expression is frequently observed in independent transgenic lines. Variability of transgene expression has been attributed to several factors, including differences in chromosome position, repeat sequences and copy number. The eukaryotic genome, with a heterogeneous chromatin structure, is not homogeneous for transcriptional activity. Chromatin structure at the site of integration can affect transgene expression; this phenomenon is called the position effect. In this study, we investigated whether position effects confer variability of transgene expression in Arabidopsis thaliana. We analyzed the expression of randomly integrated single 'complete' (intact, non-truncated, non-rearranged) copy transgenes in A. thaliana. Ten independent lines containing single complete copies of the transgene located at different chromosome positions showed very similar levels of transgene expression, and variability of transgene expression was not observed. This result indicates that position effects may not generally be a major cause of variability of transgene expression in A. thaliana.

Transcriptional activation of tobacco E2F is repressed by co-transfection with the retinoblastoma-related protein: cyclin D expression overcomes this repressor activity.

Evidence is emerging that the E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S transition in plants. Here, we show that in the tobacco proliferating cell nuclear antigen (PCNA), whose transcript is specifically expressed at G1/S phase, the two E2F binding sites are synergistically responsible for transcriptional activation at G1/S phase in synchronized tobacco BY-2 cells transformed with promoter constructs fused to a reporter gene. In addition, we have isolated the tobacco DP cDNA (NtDP) and showed that significant activation of the reporter gene was observed in transient expression assays by concomitantly transfecting with plasmids expressing NtE2F and NtDP. This transcriptional activation was repressed by co-transfection with a plasmid expressing NtRBR1; in vitro pull-down assays also revealed that NtRBR1 binds directly to NtE2F, thereby potentially blocking the transcriptional activation of NtE2F. Importantly, this repressor activity was cancelled when NtRBR1 was further co-transfected with a plasmid expressing cyclin D but not with cyclin A or cyclin B. These results are discussed with respect to the repression activity of NtRBR1 on the NtE2F/NtDP complex.

Phosphorylation of retinoblastoma-related protein by the cyclin D/cyclin-dependent kinase complex is activated at the G1/S-phase transition in tobacco.

In mammals, D-type cyclin-associated kinases mainly regulate the G1/S transition by phosphorylating the retinoblastoma (Rb) protein. We previously demonstrated that in tobacco, cyclin D (Nicta; CycD3;3) is complexed with the PSTAIRE-containing cyclin-dependent kinase (CDKA) from tobacco. Here, we show that Nicta; CycD3;3-associated kinases phosphorylate both the tobacco Rb-related protein (NtRb1) and histone H1. Although NtRb1 kinase activity was detected only during the middle G1- to early S-phase, histone H1 kinase activity was observed as two peaks in G1- to S-phase and G2/M- to M-phase. Importantly, we show that the proportion of cells in the G1-phase was reduced in transgenic Bright Yellow-2 cells overexpressing Nicta; CycD3;3-GFP. Mutational analyses revealed that phosphorylation of Thr-191 in Nicta; CycD3;3 possibly is required for both full kinase activity and localization predominantly to the nucleus. These data suggest that Nicta; CycD3;3 acts as a rate-limiting regulator in the G1/S transition by forming active complexes with CDKA or its related kinases to phosphorylate Rb-related protein and potentially plays a novel role during G2/M and mitosis.