Genetic Insulator for Preventing Influence by Another Gene Promoter

A 16 bp polynucleotide sequence of Arabidopsis thaliana is a genetic insulator that can effectively isolate a transgene from positional effects of neighboring gene activities in transgenic plant cells

Bidirectional Promoters and Methods Related Thereto

The present invention comprises artificial nucleic acid constructs comprising a bidirectional promoter having minimal promoter and a common promoter, wherein said minimal promoters is operably linked to said common promoter, in opposite orientation to said common promoter, and 5′ to said common promoter. Those artificial nucleic acid constructs, wherein said bidirectional promoter further comprises at least one gene operably linked to said minimal promoter and said common promoter are preferred

Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex

Live Imaging Study on Cytokinin Function and Regulation in Stem-cell Homeostasis

Stem-cell homeostasis is mediated by multifaceted networks involving plant hormones and local cell-cell communication. Earlier studies have implicated cytokinins in regulating shoot apical meristem (SAM) growth. However, the precise role of cytokinin in SAM remains largely unknown because cytokinins have been implicated in several developmental processes and merely studying the terminal phenotypes may not reveal their function in actively developing SAMs. The live-image technology allows studying the function of any given regulator immediately after its perturbations in transient experiments. The live-imaging work presented here provides: 1) Functional analysis of SAM-enriched cytokinin activating enzymes, 2) The live-image study on key regulators in cytokinin biosynthesis and signaling, 3) The function of SHOOTMERISTEMLESS (STM).The cell-type specific genomics has predicted and RNA in situ analysis has confirmed the enrichment of cytokinin activating enzymes in the SAM-stem cell niche. Knock-out three centrally expressed members alters phyllotaxy. Transient manipulation of cytokinin results in organ positioning defects within the peripheral zone (PZ) of SAMs. The levels of cytokinin are correlated to auxin responses. Moreover, external application of auxin fails to induce auxin responses suggesting that cytokinins are required for activating auxin response pathway components. Cell division analyses reveal that cell mitotic activities are correlated to the levels of cytokinins. We conclude that cytokinin controls phyllotaxy by regulating auxin signaling, auxin transport and cell division rates. We find that WUSCHEL (WUS) regulates a type-B response regulator, ARR1. Then, we have studied the effects of positive cytokinin signaling by using inducible activation of constitutively active forms of ARR1 coupled with live-image technology. This analysis has revealed that constitutive activation of cytokinin signaling results in expansion of stem-cell domain leading to de-differentiation of differentiating cells and this process is WUS dependent. Genetically, stm mutant phenotype implies that STM function is required for SAM initiation and fulfills a complementary role to that of WUS. Inducible inactivation of STM has shown that it is required for preventing the CZ cells from responding to auxin. Furthermore, other differentiation markers were also found to be mis-expressed in the CZ cells. These findings lead to conclude that STM prevent the stem-cell domain from differentiation

A B-ARR-mediated cytokinin transcriptional network directs hormone cross-regulation and shoot development

Cytokinin regulates gene expression by activation of ARR transcription factors. Here, the authors use ChIP-seq to show how three type B-ARRs mediate cytokinin response in Arabidopsis and provide evidence that cytokinin regulates meristem development by promoting B-ARR binding to WUSCHEL

Lower WUS protein accumulation and altered <i>CLV3</i> expression in cytokinin receptor mutants.

<p>RNA <i>in situ</i> showing localization of <i>WUS</i> transcripts in L<i>er</i> (A) and in <i>cre1-12;ahk2-2</i>;<i>ahk3-3</i> (B) lines. The number of <i>WUS</i> expressing cells quantified from RNA <i>in situ</i> images in L<i>er</i> and <i>cre1-12;ahk2-2</i>;<i>ahk3-3</i>. P>0.05 as determined by a Student’s t-test (C). WUS protein (<i>pWUS</i>::<i>eGFP-WUS</i>) accumulation in L<i>er</i> (D) and in <i>cre1-12;ahk2-2</i>;<i>ahk3-3</i> (E-F). <i>cre1-12;ahk2-2</i>;<i>ahk3-3</i> plants showing no detectable <i>pWUS</i>::<i>eGFP-WUS</i> accumulation (E), a few <i>pWUS</i>::<i>eGFP-WUS</i> accumulating cells detected when imaged at 1.5x detector gain (F). The <i>pCLV3</i>::<i>mGFP5-ER</i> expression in L<i>er</i> (G) and <i>cre1-12;ahk2-2</i>;<i>ahk3-3</i> (H). Semi-quantitative RT-PCR of <i>WUS</i> and <i>CRE1</i>/<i>AHK4</i>, <i>AHK2</i>, and <i>AHK3</i> transcripts in wild type and <i>cre1-12;ahk2-2</i>;<i>ahk3-3</i> mutants (I). The cell layers in SAMs are marked; the L1 and the L2 are monolayers. The multilayer L3 has been divided into the apical L3 layer and the basal L3 layers. The pith is located beneath the basal L3 layers. Insets for each image show the areas identified by black arrowheads at 4x zoom and white arrowheads show the boundaries of reporter accumulation. Autofluorescence is denoted by grey arrowheads and is characterized by multiple foci in a single cell. eGFP (green) is overlaid on FM4-64 (red) plasma membrane stain in (D-F). mGFP5-ER is overlaid on DIC in (G-H). The scale bars = 50 μm for all images.</p

Cytokinin stabilizes WUSCHEL by acting on the protein domains required for nuclear enrichment and transcription

<div><p>Concentration-dependent transcriptional regulation and the spatial regulation of transcription factor levels are poorly studied in plant development. WUSCHEL, a stem cell-promoting homeodomain transcription factor, accumulates at a higher level in the rib meristem than in the overlying central zone, which harbors stem cells in the shoot apical meristems of <i>Arabidopsis thaliana</i>. The differential accumulation of WUSCHEL in adjacent cells is critical for the spatial regulation and levels of <i>CLAVATA3</i>, a negative regulator of <i>WUSCHEL</i> transcription. Earlier studies have revealed that DNA-dependent dimerization, subcellular partitioning and protein destabilization control WUSCHEL protein levels and spatial accumulation. Moreover, the destabilization of WUSCHEL may also depend on the protein concentration. However, the roles of extrinsic spatial cues in maintaining differential accumulation of WUS are not understood. Through transient manipulation of hormone levels, hormone response patterns and analysis of the receptor mutants, we show that cytokinin signaling in the rib meristem acts through the transcriptional regulatory domains, the acidic domain and the WUSCHEL-box, to stabilize the WUS protein. Furthermore, we show that the same WUSCHEL-box functions as a degron sequence in cytokinin deficient regions in the central zone, leading to the destabilization of WUSCHEL. The coupled functions of the WUSCHEL-box in nuclear retention as described earlier, together with cytokinin sensing, reinforce higher nuclear accumulation of WUSCHEL in the rib meristem. In contrast a sub-threshold level may expose the WUSCHEL-box to destabilizing signals in the central zone. Thus, the cytokinin signaling acts as an asymmetric spatial cue in stabilizing the WUSCHEL protein to lead to its differential accumulation in neighboring cells, which is critical for concentration-dependent spatial regulation of <i>CLAVATA3</i> transcription and meristem maintenance. Furthermore, our work shows that cytokinin response is regulated independently of the WUSCHEL function which may provide robustness to the regulation of WUSCHEL concentration.</p></div

Cytokinin can offset ectopic WUS overexpression induced instability, which depends on the degron-like function of the WUS-box but does not require transcriptional activity of WUS.

<p>The SAMs showing the accumulation of ubiquitously expressed wild type (<i>p35S</i>::<i>eGFP-WUS-GR</i>), WUS-box mutant (<i>p35S</i>::<i>eGFP-WUS (WBM)-GR</i>), EAR-like domain mutant (<i>p35S</i>::<i>eGFP-WUS (EARLM)-GR</i>), double mutants of WBM mutant with the EARLM domain mutant (<i>p35S</i>::<i>eGFP-WUS (WBM+EARLM)-GR</i>), and double mutants of mHOD1 and ΔHOD2 (<i>p35</i>::<i>eGFP-WUS (mHOD1+ΔHOD2)-GR)</i> versions in Mock-treated (A, E, I, M, Q), Dex-treated for 24 hrs (B, F, J, N, R), 6-BAP-treated for 24 hrs (C, G, K, O, S), and combined Dex+6-BAP treated for 24 hrs (D, H, L, P, T) plants. 6-BAP treatment alone led to increase in cytoplasmically-localized WUS in wild type (C), EARLM (K), and the double mutants of mHOD1 and ΔHOD2 (S) while the WBM (G) and the double mutants of WBM and EARLM (O) did not reveal a striking increase in protein levels. Dex-treatment alone led to a dramatically lower levels of protein accumulation in cells located in the SAM (shown by the black arrowhead) in comparison to cells located in the developing leaves and the pith (yellow arrowheads) in wild type (B) and the double mutants of mHOD1 and ΔHOD2 (R) while the combined Dex and 6-BAP treatment improved protein accumulation both in wild type (D) and the double mutants of mHOD1 and ΔHOD2 (T). The Dex treatment of the WBM (F), EARLM (J) and the double mutants of WBM and EARLM (N) led to stable nuclear accumulation, though the WBM (F) did not show tight nuclear localization seen with the EARLM (J) and the WBM plus EARLM double mutants (N). The combined Dex and 6-BAP treatment (H, L, P) did not reveal a striking difference from that of the Dex alone treatment (F, J, N) in all three WUS forms. The cell layers in SAMs are marked; the L1 and the L2 are monolayers. The multilayer L3 has been divided into the apical L3 layer and the basal L3 layers. The pith is located beneath the basal L3 layers. Insets for each image show the areas identified by black arrowheads at 4x zoom. eGFP (green) is overlaid on FM4-64 (red) plasma membrane stain. The scale bars = 50 μm for all images.</p