Processes In Arabidopsis Research Articles

Function of the Golgi-located phosphate transporter PHT4;6 is critical for senescence-associated processes in Arabidopsis.

The phosphate transporter PHT4;6 locates to the trans-Golgi compartment, and its impaired activity causes altered intracellular phosphate compartmentation, leading to low cytosolic Pi levels, a blockage of Golgi-related processes such as protein glycosylation and hemicellulose biosynthesis, and a dwarf phenotype. However, it was unclear whether altered Pi homeostasis in pht4;6 mutants causes further cellular problems, typically associated with limited phosphate availability. Here we report that pht4;6 mutants exhibit a markedly increased disposition to induce dark-induced senescence. In control experiments, in which pht4;6 mutants and wild-type plants developed similarly, we confirmed that accelerated dark-induced senescence in mutants is not a 'pleiotropic' process associated with the dwarf phenotype. In fact, accelerated dark-induced senescence in pht4;6 mutants correlates strongly with increased levels of toxic NH4 (+) and higher sensitivity to ammonium, which probably contribute to the inability of pht4;6 mutants to recover from dark treatment. Experiments with modified levels of either salicylic acid (SA) or trans-zeatin (tZ) demonstrate that altered concentrations of these compounds in pht4;6 plants act as major cellular mediators for dark-induced senescence. This conclusion gained further support from the notion that the expression of the pht4;6 gene is, in contrast to genes coding for major phosphate importers, substantially induced by tZ. Taken together, our findings point to a critical function of PHT4;6 to control cellular phosphate levels, in particular the cytosolic Pi availability, required to energize plant primary metabolism for proper plant development. Phosphate and its allocation mediated by PHT4;6 is critical to prevent onset of dark-induced senescence.

Genome-Wide Analysis of Gene Regulatory Networks of the FVE-HDA6-FLD Complex in Arabidopsis.

FVE/MSI4 is a homolog of the mammalian RbAp48 protein. We found that FVE regulates flowering time by repressing FLC through decreasing histone H3K4 trimethylation and H3 acetylation. Furthermore, FVE interacts with the histone deacetylase HDA6 and the histone demethylase FLD, suggesting that these proteins may form a protein complex to regulate flowering time. To further investigate the function of the FVE-FLD-HDA6 complex, we compared the gene expression profiles of fve, fld, and hda6 mutant plants by using RNA-seq analysis. Among the mis-regulated genes found in fve plants, 51.8 and 36.5% of them were also mis-regulated in fld and hda6 plants, respectively, suggesting that FVE, HDA6, and FLD may regulate the gene expression in the same developmental processes in Arabidopsis. Gene ontology analysis revealed that among 383 genes co-regulated by FVE, HDA6, and FLD, 15.6% of them are involved in transcription, 8.2% in RNA metabolic process, 7.7% in response to abiotic stress, and 6.3% in hormone stimulus. Taken together, these results indicate that HDA6, FVE, and FLD co-regulate the gene expression in multiple development processes and pathways.

CHD3 chromatin-remodeling factor PICKLE regulates floral transition partially via modulating LEAFY expression at the chromatin level in Arabidopsis.

PICKLE (PKL), a putative CHD3 chromatin remodeling factor, has been suggested to be involved in multiple processes in Arabidopsis. Here, we confirmed the late-flowering phenotype caused by pkl mutation with pkl mutants in two different ecotypes, and investigated the possible mechanisms that account for PKL regulation of flowering time. Quantitative RT-PCR and RNA-seq assays showed that expression of the LEAFY gene (LFY) and a number of LFY-regulated floral homeotic genes were down-regulated in seedlings of the pkl mutants. As predicted, overexpression of LFY restored normal flowering time of pkl mutants. Our results suggest that PKL may be involved in regulating flowering time via LFY expression. To uncover the underlying mechanism, ChIP-PCR using anti-PKL was performed on materials from three developmental stages of seedlings. Our results showed that PKL associated with the genomic sequences of LFY, particularly at 10-day and 25-day after germination. We also showed that loss of PKL affected H3K27me3 level at the promoter of LFY. Taken together, our data suggest that transcriptional regulation of LFY at the chromatin level by PKL may at least partially account for the late-flowering phenotype of pkl mutants.

A mutation of casein kinase 2 α4 subunit affects multiple developmental processes in Arabidopsis.

Arabidopsis CK2 α4 subunit regulates the primary root and hypocotyl elongation, lateral root formation, cotyledon expansion, rosette leaf initiation and growth, flowering, and anthocyanin biosynthesis. Casein kinase 2 (CK2) is a conserved tetrameric kinase composed of two α and two β subunits. The inhibition of CK2 activity usually results in severe developmental deficiency. Four genes (CKA1-CKA4) encode CK2 α subunit in Arabidopsis. Single mutations of CKA1, CKA2, and CKA3 do not affect the normal growth of Arabidopsis, while the cka1 cka2 cka3 triple mutants are defective in cotyledon and hypocotyl growth, lateral root development, and flowering. The inhibition of CKA4 expression in cka1 cka2 cka3 background further reduces the number of lateral roots and delays the flowering time. Here, we report the characterization of a novel knockout mutant of CKA4, which exhibits various developmental defects including reduced primary root and hypocotyl elongation, increased lateral root density, delayed cotyledon expansion, retarded rosette leaf initiation and growth, and late flowering. The examination of the cellular basis for abnormal root development of this mutant revealed reduced root meristem cells with enhanced RETINOBLASTOMA-RELATED (RBR) expression that promotes cell differentiation in root meristem. Moreover, this cka4-2 mutant accumulates higher anthocyanin in the aerial part and shows an increased expression of anthocyanin biosynthetic genes, suggesting a novel role of CK2 in modulating anthocyanin biosynthesis. In addition, the complementation test using primary root elongation assay as a sample confirms that the changed phenotypes of this cka4-2 mutant are due to the lack of CKA4. Taken together, this study reveals an essential role of CK2 α4 subunit in multiple developmental processes in Arabidopsis.

Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association.

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2’s repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition.

High-resolution mapping of H4K16 and H3K23 acetylation reveals conserved and unique distribution patterns in Arabidopsis and rice

Histone acetylation and deacetylation are key epigenetic gene regulatory mechanisms that play critical roles in eukaryotes. Acetylation of histone 4 lysine 16 (H4K16ac) is implicated in many cellular processes. However, its biological function and relationship with transcription are largely unexplored in plants. We generated first genome-wide high-resolution maps of H4K16ac in Arabidopsis thaliana and Oryza sativa. We showed that H4K16ac is preferentially enriched around the transcription start sites and positively correlates with gene expression levels. Co-existence of H4K16ac and H3K23ac is correlated with high gene expression levels, suggesting a potentially combinatorial effect of H4K16ac and H3K23ac histone 3 lysine 23 acetylation on gene expression. Our data further revealed that while genes enriched with both H4K16ac and H3K23ac are ubiquitously expressed, genes enriched with only H4K16ac or H3K23ac showed significantly distinct expression patterns in association with particular developmental stages. Unexpectedly, and unlike in Arabidopsis, there are significant levels of both H4K16ac and H3K23ac in the lowly expressed genes in rice. Furthermore, we found that H4K16ac-enriched genes are associated with different biological processes in Arabidopsis and rice, suggesting a potentially species-specific role of H4K16ac in plants. Together, our genome-wide profiling reveals the conserved and unique distribution patterns of H4K16ac and H3K23ac in Arabidopsis and rice and provides a foundation for further understanding their function in plants.

A Conserved Core of Programmed Cell Death Indicator Genes Discriminates Developmentally and Environmentally Induced Programmed Cell Death in Plants.

A plethora of diverse programmed cell death (PCD) processes has been described in living organisms. In animals and plants, different forms of PCD play crucial roles in development, immunity, and responses to the environment. While the molecular control of some animal PCD forms such as apoptosis is known in great detail, we still know comparatively little about the regulation of the diverse types of plant PCD. In part, this deficiency in molecular understanding is caused by the lack of reliable reporters to detect PCD processes. Here, we addressed this issue by using a combination of bioinformatics approaches to identify commonly regulated genes during diverse plant PCD processes in Arabidopsis (Arabidopsis thaliana). Our results indicate that the transcriptional signatures of developmentally controlled cell death are largely distinct from the ones associated with environmentally induced cell death. Moreover, different cases of developmental PCD share a set of cell death-associated genes. Most of these genes are evolutionary conserved within the green plant lineage, arguing for an evolutionary conserved core machinery of developmental PCD. Based on this information, we established an array of specific promoter-reporter lines for developmental PCD in Arabidopsis. These PCD indicators represent a powerful resource that can be used in addition to established morphological and biochemical methods to detect and analyze PCD processes in vivo and in planta.

Functional characterization of Ostreococcus tauri phototropin.

Phototropins (phots) regulate a range of adaptive processes in plants that serve to optimize photosynthetic efficiency and promote growth. Light sensing by Arabidopsis thaliana phots is predominantly mediated by the Light, Oxygen and Voltage sensing 2 (LOV2) flavin-binding motif located within the N-terminus of the photoreceptor. Here we characterize the photochemical and biochemical properties of phot from the marine picoalga Ostreococcus tauri phototropin (Otphot) and examine its ability to replace phot-mediated function in Arabidopsis. Photochemical properties of Otphot rely on both LOV1 and LOV2. Yet, biochemical analysis indicates that light-dependent receptor autophosphorylation is primarily dependent on LOV2. As found for Arabidopsis phots, Otphot associates with the plasma membrane and partially internalizes, albeit to a limited extent, in response to blue-light irradiation. Otphot is able to elicit a number of phot-regulated processes in Arabidopsis, including petiole positioning, leaf expansion, stomatal opening and chloroplast accumulation movement. However, Otphot is unable to restore phototropism and chloroplast avoidance movement. Consistent with its lack of phototropic function in Arabidopsis, Otphot does not associate with or trigger dephosphorylation of the phototropic signalling component Non-Phototropic Hypocotyl 3 (NPH3). Taken together, these findings indicate that the mechanism of action of plant and evolutionarily distant algal phots is less well conserved than previously thought.

Expression profiling and functional analysis reveals that TOR is a key player in regulating photosynthesis and phytohormone signaling pathways in Arabidopsis.

Target of rapamycin (TOR) acts as a master regulator to control cell growth by integrating nutrient, energy, and growth factors in all eukaryotic species. TOR plays an evolutionarily conserved role in regulating the transcription of genes associated with anabolic and catabolic processes in Arabidopsis, but little is known about the functions of TOR in photosynthesis and phytohormone signaling, which are unique features of plants. In this study, AZD8055 (AZD) was screened as the strongest active-site TOR inhibitor (asTORi) in Arabidopsis compared with TORIN1 and KU63794 (KU). Gene expression profiles were evaluated using RNA-seq after treating Arabidopsis seedlings with AZD. More than three-fold differentially expressed genes (DEGs) were identified in AZD-treated plants relative to rapamycin-treated plants in previous studies. Most of the DEGs and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways involved in cell wall elongation, ribosome biogenesis, and cell autophagy were common to both AZD- and rapamycin-treated samples, but AZD displayed much broader and more efficient inhibition of TOR compared with rapamycin. Importantly, the suppression of TOR by AZD resulted in remodeling of the expression profile of the genes associated with photosynthesis and various phytohormones, indicating that TOR plays a crucial role in modulating photosynthesis and phytohormone signaling in Arabidopsis. These newly identified DEGs expand the understanding of TOR signaling in plants. This study elucidates the novel functions of TOR in photosynthesis and phytohormone signaling and provides a platform to study the downstream targets of TOR in Arabidopsis.

Three SAUR proteins SAUR76, SAUR77 and SAUR78 promote plant growth in Arabidopsis.

Ethylene perceived by a family of five receptors regulates many developmental processes in Arabidopsis. Here we conducted the yeast two-hybrid assay to screen for additional unidentified proteins that interact with subfamily II ethylene receptor ETR2. Three SAUR proteins, named SAUR76, 77 and 78, were identified to associate with both ETR2 and EIN4 in different assays. Interaction of SAUR76 and SAUR78 with ETR2 was further verified by co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays. Expressions of SAUR76-78 are induced by auxin and ethylene treatments. Compared with wild type, SAUR-overexpressing plants exhibit reduced ethylene sensitivity, while SAUR-RNAi lines exhibit enhanced ethylene sensitivity. Overexpressing the three SAURs partially complements the phenotype of subfamily II ethylene receptor loss-of-function double mutant etr2-3ein4-4, which has increased ethylene response and small cotyledon and rosette. saur76 mutation partially suppresses the reduced ethylene sensitivity of etr2-2. SAUR76/78 proteins are regulated by 26S proteasome system and larger tag increases their protein stability. These findings suggest that SAUR76-78 may affect ethylene receptor signaling and promote plant growth in Arabidopsis.

The ULTRAPETALA1 trxG factor contributes to patterning the Arabidopsis adaxial-abaxial leaf polarity axis

The SAND domain protein ULTRAPETALA1 (ULT1) functions as a trithorax group factor that regulates a variety of developmental processes in Arabidopsis. We have recently shown that ULT1 regulates developmental patterning in the gynoecia and leaves. ULT1 acts together with the KANADI1 (KAN1) transcription factor to pattern the apical-basal axis during gynoecium formation, whereas the 2 genes act antagonistically to pattern the adaxial-abaxial axis during both gynoecium and leaf formation. In particular, our data showed that ULT1 is necessary for the kan1 adaxialized organ phenotype. Here, we observe the internal structure of ult1, kan1 and ult1 kan1 rosette leaves to better understand the suppression of the kan1 adaxialized leaf polarity defect by ult1 mutations. Our results indicate that ULT1 and KAN1 act antagonistically to pattern the adaxial-abaxial axis in leaves by establishing the asymmetry of the internal cell layers.

Characterization of xanthophyll pigments, photosynthetic performance, photon energy dissipation, reactive oxygen species generation and carbon isotope discrimination during artemisinin-induced stress in Arabidopsis thaliana.

Artemisinin, a potent antimalarial drug, is phytotoxic to many crops and weeds. The effects of artemisinin on stress markers, including fluorescence parameters, photosystem II photochemistry, photon energy dissipation, lipid peroxidation, reactive oxygen species generation and carbon isotope discrimination in Arabidopsis thaliana were studied. Arabidopsis ecotype Columbia (Col-0) seedlings were grown in perlite and watered with 50% Hoagland nutrient solution. Adult plants of Arabidopsis were treated with artemisinin at 0, 40, 80, 160 μM for one week. Artemisinin, in the range 40–160 μM, decreased the fresh biomass, chl a, b and leaf mineral contents. Photosynthetic efficiency, yield and electron transport rate in Arabidopsis were also reduced following exposure to 80 and 160 μM artemisinin. The ΦNPQ and NPQ were less than control. Artemisinin treatment caused an increase in root oxidizability and lipid peroxidation (MDA contents) of Arabidopsis. Calcium and nitrogen contents decreased after 80 and 160 μM artemisinin treatment compared to control. δ13C values were less negative following treatment with artemisinin as compared to the control. Artemisinin also decreased leaf protein contents in Arabidopsis. Taken together, these data suggest that artemisinin inhibits many physiological and biochemical processes in Arabidopsis.

Arabidopsis DOK1 encodes a functional dolichol kinase involved in reproduction.

Dolichol phosphate (Dol-P) serves as a carrier of complex polysaccharides during protein glycosylation. Dol-P is synthesized by the phosphorylation of dolichol or the monodephosphorylation of dolichol pyrophosphate (Dol-PP); however, the enzymes that catalyze these reactions remain unidentified in Arabidopsis thaliana. We performed a genome-wide search for cytidylyltransferase motif-containing proteins in Arabidopsis, and found that At3g45040 encodes a protein homologous with Sec59p, a dolichol kinase (DOK) in Saccharomyces cerevisiae. At3g45040, designated AtDOK1, complemented defects in the growth and N-linked glycosylation of the S.cerevisiae sec59 mutant, suggesting that AtDOK1 encodes a functional DOK. To characterize the physiological roles of AtDOK1 in planta, we isolated two independent lines of T-DNA-tagged AtDOK1 mutants, dok1-1 and dok1-2. The heterozygous plants showed developmental defects in male and female gametophytes, including an aberrant pollen structure, low pollen viability, and short siliques. Additionally, the mutations had incomplete penetrance. These results suggest that AtDOK1 is a functional DOK required for reproductive processes in Arabidopsis.

Meta-analysis of gene expression signatures reveals hidden links among diverse biological processes in Arabidopsis.

The model plant Arabidopsis has been well-studied using high-throughput genomics technologies, which usually generate lists of differentially expressed genes under various conditions. Our group recently collected 1065 gene lists from 397 gene expression studies as a knowledgebase for pathway analysis. Here we systematically analyzed these gene lists by computing overlaps in all-vs.-all comparisons. We identified 16,261 statistically significant overlaps, represented by an undirected network in which nodes correspond to gene lists and edges indicate significant overlaps. The network highlights the correlation across the gene expression signatures of the diverse biological processes. We also partitioned the main network into 20 sub-networks, representing groups of highly similar expression signatures. These are common sets of genes that were co-regulated under different treatments or conditions and are often related to specific biological themes. Overall, our result suggests that diverse gene expression signatures are highly interconnected in a modular fashion.

Characterization of Arabidopsis Tubby-like proteins and redundant function of AtTLP3 and AtTLP9 in plant response to ABA and osmotic stress.

Tubby and Tubby-like proteins (TLPs) play essential roles in the development and function of mammal neuronal cells. In addition to the conserved carboxyl (C)-terminal Tubby domain, which is required for their plasma membrane (PM) tethering, plant TLPs also possess an amino (N)-terminal F-box domain to interact with specific Arabidopsis Skp1-like (ASK) proteins as functional SCF-type E3 ligases. Here, we report the molecular characterization of Arabidopsis TLPs (AtTLPs). β-Glucuronidase staining showed overlapped but distinct expression patterns of AtTLPs in Arabidopsis. Yeast two-hybrid assays further revealed that AtTLP1, AtTLP3, AtTLP6, AtTLP7, AtTLP9, AtTLP10 and AtTLP11 all interacted with specific ASKs, but AtTLP2, AtTLP5 and AtTLP8 did not. Subcellular localization observations in both Arabidopsis protoplasts and tobacco pollen tubes indicated that all GFP-AtTLP fusion proteins, except GFP-AtTLP8 which lacks the conserved phosphatidylinositol 4,5-bisphosphate binding sites, were targeted to the PM. Detailed studies on AtTLP3 demonstrated that AtTLP3 is a PM-tethered PIP2 binding protein which functions redundantly with AtTLP9 in abscisic acid (ABA)- and osmotic stress-mediated seed germination. Our results suggest that AtTLPs possibly work in multiple physiological and developmental processes in Arabidopsis, and AtTLP3 is also involved in ABA signaling pathway like AtTLP9 during seed germination and early seedling growth.

Arabidopsis Retinoblastoma-related and Polycomb group proteins: cooperation during plant cell differentiation and development

RETINOBLASTOMA (RB) is a tumour suppressor gene originally discovered in patients that develop eye tumours. The pRb protein is now well established as a key cell-cycle regulator which suppresses G1-S transition via interaction with E2F-DP complexes. pRb function is also required for a wide range of biological processes, including the regulation of stem-cell maintenance, cell differentiation, permanent cell-cycle exit, DNA repair, and genome stability. Such multifunctionality of pRb is thought to be facilitated through interactions with various binding partners in a context-dependent manner. Although the molecular network in which RB controls various biological processes is not fully understood, it has been found that pRb interacts with transcription factors and chromatin modifiers to either suppress or promote the expression of key genes during the switch from cell proliferation to differentiation. RETINOBLASTOMA-RELATED (RBR) is the plant orthologue of RB and is also known to negatively control the G1-S transition. Similar to its animal counterpart, plant RBR has various roles throughout plant development; however, much of its molecular functions outside of the G1-S transition are still unknown. One of the better-characterized molecular mechanisms is the cooperation of RBR with the Polycomb repressive complex 2 (PRC2) during plant-specific developmental events. This review summarizes the current understanding of this cooperation and focuses on the processes in Arabidopsis in which the RBR-PRC2 cooperation facilitates cell differentiation and developmental transitions.

Identification of SUMO-modified proteins by affinity purification and tandem mass spectrometry in Arabidopsis thaliana

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in eukaryotes. Although more than 200 SUMO target proteins have been characterized in yeasts and animals, only very few SUMO targets are known in plants. Here, to identify putative SUM1 associating proteins in Arabidopsis we developed a proteomics-type protocol employing a His-GST-AtSUM1 column conjugated to Affi-gel beads with total protein extracts from 3–4 week old seedlings. AtSUM1 binding proteins and/or sumoylated targets were obtained by a change in pH of the eluant and identified by mass spectrometry. Fifteen candidate SUMO-modified proteins have so far been identified. Candidate proteins were analyzed by a split ubiquitination assay in yeast and additionally by split-luciferase complement assays in planta to confirm binding specificity and activity to AtSUM1, and productive sumoylation through in vivo assays in E. coli. Among proteins found reliably sumoylated are AtMYB31 (transcription factor), ACS7 (1-amino-cyclopropane-1-carboxylate synthase 7), and Rho GDI1 (Rho GDPdissociation inhibitor family protein). These proteins will provide a basis for studying functions of SUMOylation in the regulation of diverse processes in Arabidopsis.

Proteome Analysis in Arabidopsis Reveals Shoot- and Root-Specific Targets of Cytokinin Action and Differential Regulation of Hormonal Homeostasis

The plant hormones cytokinins (CKs) regulate multiple developmental and physiological processes in Arabidopsis (Arabidopsis thaliana). Responses to CKs vary in different organs and tissues (e.g. the response to CKs has been shown to be opposite in shoot and root samples). However, the tissue-specific targets of CKs and the mechanisms underlying such specificity remain largely unclear. Here, we show that the Arabidopsis proteome responds with strong tissue and time specificity to the aromatic CK 6-benzylaminopurine (BAP) and that fast posttranscriptional and/or posttranslational regulation of protein abundance is involved in the contrasting shoot and root proteome responses to BAP. We demonstrate that BAP predominantly regulates proteins involved in carbohydrate and energy metabolism in the shoot as well as protein synthesis and destination in the root. Furthermore, we found that BAP treatment affects endogenous hormonal homeostasis, again with strong tissue specificity. In the shoot, BAP up-regulates the abundance of proteins involved in abscisic acid (ABA) biosynthesis and the ABA response, whereas in the root, BAP rapidly and strongly up-regulates the majority of proteins in the ethylene biosynthetic pathway. This was further corroborated by direct measurements of hormone metabolites, showing that BAP increases ABA levels in the shoot and 1-aminocyclopropane-1-carboxylic acid, the rate-limiting precursor of ethylene biosynthesis, in the root. In support of the physiological importance of these findings, we identified the role of proteins mediating BAP-induced ethylene production, METHIONINE SYNTHASE1 and ACC OXIDASE2, in the early root growth response to BAP.

Functional identification of AtSKIP as a regulator of the cell cycle signaling pathway in Arabidopsis thaliana

Light is a key environmental cue controlling plant development, which involves meristemic activation by cell proliferation and differentiation. Here, we identify one gene, AtSKIP, associated with cell cycle-regulated root and leaf growth processes in Arabidopsis. The spatial pattern of β-glucuronidase (GUS) activity indicated that AtSKIP is expressed in the leaf primodia, root meristem region and root vascular system, and can be activated by light. Ectopic expression of AtSKIP resulted in enhanced leaf development but suppressed root elongation in Arabidopsis, whereas AtSKIPDD seedlings displayed retarded leaf growth and normal root growth. Moreover, AtSKIP cells displayed enhanced sensitivity to a cytokinin in a callus induction assay, further demonstrated that AtSKIP expression altered endogenous cell cycle-regulated signaling in plants. Together, these data indicate that AtSKIP participates in cell cycle-mediated growth of leaf and root.

Expression, purification and analysis of an Arabidopsis recombinant CBL-interacting protein kinase3 (CIPK3) and its constitutively active form

CIPK3 is a member of CBL (calcineurin B-like)-interacting serine–threonine protein kinases which play an important role in many developmental and adaptation processes in Arabidopsis. Studies conducted on members of this family such as SOS2, PKS8 and PKS11 have provided insight into how these kinases interact with their target substrates in the signal-response process. Since SOS2, PKS8 and PKS11 have low enzymatic activities in vitro and their amino acid sequences are homologous to that of CIPK3, it was assumed that CIPK3 would have a low enzymatic activity. To enhance CIPK3 enzyme activity, a constitutively active form, CIPK3T183D, was generated by a Thr183 to Asp183 substitution in the activation loop. To obtain proteins for analysis, glutathione S-transferase (GST) fusion protein system was used. Although both CIPK3 and CIPK3T183D were successfully expressed, they were found in inclusion bodies with three truncated proteins. Since the truncated proteins had a similar affinity to the GST-Bind Resin as the target protein, the one-step affinity purification could no longer be used. As an alternative, His fusion protein expression system was employed for protein production. Although both His-CIPK3 and His-CIPK3T183D also accumulated in inclusion bodies, they were expressed as a single protein species. A method involving Sarkosyl was developed for isolating and purifying the His fusion proteins. His-CIPK3 and His-CIPK3T183D produced were highly purified and enzymatically active. In addition, a 9-fold increase in kinase activity in His-CIPK3T183D was observed, indicating that Thr183 to Asp183 substitution in the activation loop of CIPK3 had succeeded in enhancing the kinase activity.