Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity

Synchronized suspension cultures are powerful tools in plant cell-cycle studies. However, few Arabidopsis cell cultures are available, and synchrony extending over several sequential phases of the cell cycle has not been reported. Here we describe the first useful synchrony in Arabidopsis, achieved by selecting the rapidly dividing Arabidopsis cell suspensions MM1 and MM2d. Synchrony may be achieved either by removing and re-supplying sucrose to the growth media or by applying an aphidicolin block/release. Synchronization with aphidicolin produced up to 80% S-phase cells and up to 92% G2 cells, together with clear separation of different cell-cycle phases. These synchronization procedures can be used for analysis of gene expression and protein activity. We show that representatives of three CDK gene classes of Arabidopsis (CDKA, CDKB1 and CDKB2) show differential expression timing, and that three CDK inhibitor genes show strikingly different expression patterns during cell-cycle re-entry. We propose that ICK2 (KRP2) may have a specific role in this process.     

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.     

Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes

Growth of tomato fruits is determined by cell division and cell expansion, which are tightly controlled by factors that drive the core cell cycle. The cyclin-dependent kinases (CDKs) and their interacting partners, the cyclins, play a key role in the progression of the cell cycle. In this study the role of CDKA1, CDKB1, and CDKB2 in fruit development was characterized by fruit-specific overexpression and down-regulation. CDKA1 is expressed in the pericarp throughout development, but is strongly up-regulated in the outer pericarp cell layers at the end of the growth period, when CDKB gene expression has ceased. Overexpression of the CDKB genes at later stages of development and the down-regulation of CDKA1 result in a very similar fruit phenotype, showing a reduction in the number of cell layers in the pericarp and alterations in the desiccation of the fruits. Expression studies revealed that CDKA1 is down-regulated by the expression of CDKB1/2 in CDKB1 and CDKB2 overexpression mutants, suggesting opposite roles for these types of CDK proteins in tomato pericarp development.     

Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization

Endosperm development in maize (Zea mays L.) and related cereals comprises a cell proliferation stage followed by a period of rapid growth coupled to endoreduplication. Regulation of the cell cycle in developing endosperm is poorly understood. We have characterized various subunits of cyclin-dependent kinase (CDK) complexes, master cell cycle regulators in all eukaryotes. A-, B-, and D-type cyclins as well as A- and B-type cyclin-dependent kinases were characterized with respect to their RNA and protein expression profiles. Two main patterns were identified: one showing expression throughout endosperm development, and another characterized by a sharp down-regulation with the onset of endoreduplication. Cyclin CYCB1;3 and CYCD2;1 proteins were distributed in the cytoplasm and nucleus of cells throughout the endosperm, while cyclin CYCD5 protein was localized in the cytoplasm of peripheral cells. CDKB1;1 expression was strongly associated with cell proliferation. Expression and cyclin-binding patterns suggested that CDKA;1 and CDKA;3 are at least partially redundant. The kinase activity associated with the cyclin CYCA1 was highest during the mitotic stage of development, while that associated with CYCB1;3, CYCD2;1 and CYCD5 peaked at the mitosis-to-endoreduplication transition. A-, B- and D-type cyclins were more resistant to proteasome-dependent degradation in endoreduplicating than in mitotic endosperm extracts. These results indicated that endosperm development is characterized by differential expression and activity of specific cyclins and CDKs, and suggested that endoreduplication is associated with reduced cyclin proteolysis via the ubiquitin–proteasome pathway.     

Natural Synchronisation for the Study of Cell Division in the Green Unicellular Alga Ostreococcus tauri

Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.     

A distinct type of cyclin D, CYCD4;2, involved in the activation of cell division in Arabidopsis

The Arabidopsis genome encodes 10 D-type cyclins (CYCD); however, their differential role in cell cycle control is not well known. Among them, CYCD4;2 is unique in the amino acid sequence; namely, it lacks the Rb-binding motif and the PEST sequence that are conserved in CYCDs. Here, we have shown that CYCD4;2 suppressed G1 cyclin mutations in yeast and formed a kinase complex with CDKA;1, an ortholog of yeast Cdc28, in insect cells. Hypocotyl explants of CYCD4;2 over-expressing plants showed faster induction of calli than wild-type explants on a medium containing lower concentration of auxin. These results suggest that CYCD4;2 has a promotive function in cell division by interacting with CDKA;1 regardless of the unusual primary sequence.     

Modulation of CycD3;1‐CDK complexes by phytohormones and sucrose during maize germination

Maize CycD3;1 associates to CDKA or CDKB1;1 proteins during germination and the complexes formed develop kinase activity. These complexes appear to vary in size as germination proceeds, suggesting association to different sets of proteins. CycD3;1 and associated CDK proteins respond to phytohormones and sucrose. Results revealed a reduction in the CycD3;1 protein amount along germination in the presence of indoleacetic acid (IAA) or abscisic acid (ABA), although in the latter protein levels recover at the end of germination. While the levels of CDKA increase with IAA, they decrease with ABA. Both phytohormones, IAA and ABA, increase levels of CDKB1;1 only during the early germination times. CycD3;1 associated kinase activity is only reduced by both phytohormones towards the end of the germination period. On the other hand, lack of sucrose in the imbibition medium strongly reduces CycD3;1 protein levels without affecting the levels of neither CDKA nor CDKB1;1. The corresponding CycD3;1 associated kinase activity is also severely decreased. The presence of sucrose in the medium appears to stabilize the CycD3;1 protein levels.     

A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom

Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage.     

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40?°C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200?g?L(-1)) at 40?°C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2?g?L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7?%, respectively. In the range of 30 to 40?°C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.     

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Aims: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. Methods and Results: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase‐producing yeast Kluyveromyces marxianus Y179 and fed‐batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch‐based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l^?1 was achieved when 0·15 g l^?1 K₂HPO₄ was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. Conclusions: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Significance and Impact of the Study: Jerusalem artichoke tubers are an alternative to grain‐based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.     

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater

Aims: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. Methods and Results: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38°C for growth and inulinase production, and 35°C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l^?1, and the highest ethanol yield of 0·467, or 91·5% of the theoretical value of 0·511, was achieved for the slurry with a solid concentration of 200 g l^?1. Conclusions: Halophilic Jerusalem artichoke can be used for fuel ethanol production. Significance and Impact of the Study: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.