Project description:In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified.
Project description:In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified. 10-day-old 35S::FLAG-GR-rREV plants were treated with 6-benzylaminopurine (6-BA), dexamethasone (DEX), or 6-BA+DEX for 2 hours. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. Total RNA was extracted with RNeasy Mini Kit and hybridized to Agilent Arabidopsis Gene Expression Microarray. Differentially expressed genes were defined by a 1.5-fold expression difference with a P value<0.05. Biological replicates were performed.
Project description:Results from experiments done on knock-down of adaxial class III HD ZIP genes, using overexpression of miR165a, in shoot meristem show that Class III HD-ZIP genes act generally to repress the formation of new growth axes where they are expressed. This study explored the genes directly regualted by miRNAs as well as indirectly regulated by class III HD-ZIPs.
Project description:The class III HD-ZIP transcription factors regulate vascular patterning in Arabidopsis thaliana roots. In this expression study we compare the expression profile of the athb8 cna phb phv quadruple mutant to its wild type. The results are presented in PHABULOSA mediates an auxin signaling loop to regulate vascular patterning in Arabidopsis by Christina Joy Müller, Ana Elisa Valdés, Guodong Wang, Prashanth Ramachandran, Lisa Beste, Daniel Uddenberg, and Annelie Carlsbecker, accepted for publication in Plant Physiology Nov. 2015. Plant vascular tissues, xylem and phloem, differentiate in distinct patterns from procambial cells as an integral transport system for water, sugars and signaling molecules. Procambium formation is promoted by high auxin levels activating class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs). In the root of Arabidopsis thaliana, HD-ZIP III TFs dose-dependently govern the patterning of the xylem axis, with higher levels promoting metaxylem cell identity in the central axis and lower levels protoxylem at its flanks. It is, however, unclear by what mechanisms the HD-ZIP III TFs control xylem axis patterning. Here we present data suggesting that an important mechanism is their ability to moderate auxin response. We found that changes in HD-ZIP III TF levels affect the expression of genes encoding core auxin response molecules. We show that one of the HD-ZIP III TFs, PHABULOSA, directly binds the promoter of both MONOPTEROS/AUXIN RESPONSE FACTOR5 (MP/ARF5), a key factor in vascular formation, and IAA20, encoding an AUX/IAA protein which is stable in the presence of auxin and able to interact with and repress MP activity. The double mutant of IAA20 and its closest homologue IAA30 forms ectopic protoxylem, while overexpression of IAA30 causes discontinuous protoxylem and occasional ectopic metaxylem, similar to a weak loss-of-function mp-mutant. Our results provide evidence that HD-ZIP III TFs directly affect auxin response and mediate a feed forward loop formed by MP and IAA20 that may focus and stabilize auxin response during vascular patterning and differentiation of xylem cell types.
Project description:In addition to apical growth, plants undergo radial growth to increase girth, a particularly prominent feature in trees. During both apical and radial growth the phytohormones auxin and cytokinins form symmetries that govern further growth patterns. But whereas the gene regulatory networks interpreting the hormonal fields during apical growth are well established, such networks are not known for the radial growth. We show here that the initiation of radial growth occurs around early protophloem sieve element (PSE) cell files of the root procambial tissue in Arabidopsis. In this domain cytokinin signalling promotes expression of a pair of novel mobile transcription factors, PHLOEM EARLY DOF (PEAR1, PEAR2) and their four homologs (OBP2, DOF6, TMO6 and HCA2), collectively called PEAR proteins. The PEAR proteins form a short-range concentration gradient peaking at PSE and activating gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by well-established polarity transcription factors, HD-ZIP III, whose expression is concentrated in the more internal domain of radially non-dividing procambial cells by the function of auxin and mobile miR165/166. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, thereby establishing a negative feedback loop that forms a sharp boundary demarking the zone of cell divisions. Taken together, we have established a network, in which the PEAR - HD-ZIP III module integrates the spatial information of the hormonal domains and miRNA gradients during root procambial development, to provide a pre-pattern with actively dividing and more quiescent zones, thus priming radial growth.
Project description:The class III HD-ZIP transcription factors regulate vascular patterning in Arabidopsis thaliana roots. In this expression study we compare the expression profile of the cna-2 phb-13 phv-11 and cna-2 phb-13 phv-11 rev-6 mutants to their wild type. The results are presented in PHABULOSA mediates an auxin signaling loop to regulate vascular patterning in Arabidopsis by Christina Joy Müller, Ana Elisa Valdés, Guodong Wang, Prashanth Ramachandran, Lisa Beste, Daniel Uddenberg, and Annelie Carlsbecker, accepted for publication in Plant Physiology Nov. 2015. Plant vascular tissues, xylem and phloem, differentiate in distinct patterns from procambial cells as an integral transport system for water, sugars and signaling molecules. Procambium formation is promoted by high auxin levels activating class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs). In the root of Arabidopsis thaliana, HD-ZIP III TFs dose-dependently govern the patterning of the xylem axis, with higher levels promoting metaxylem cell identity in the central axis and lower levels protoxylem at its flanks. It is, however, unclear by what mechanisms the HD-ZIP III TFs control xylem axis patterning. Here we present data suggesting that an important mechanism is their ability to moderate auxin response. We found that changes in HD-ZIP III TF levels affect the expression of genes encoding core auxin response molecules. We show that one of the HD-ZIP III TFs, PHABULOSA, directly binds the promoter of both MONOPTEROS/AUXIN RESPONSE FACTOR5 (MP/ARF5), a key factor in vascular formation, and IAA20, encoding an AUX/IAA protein which is stable in the presence of auxin and able to interact with and repress MP activity. The double mutant of IAA20 and its closest homologue IAA30 forms ectopic protoxylem, while overexpression of IAA30 causes discontinuous protoxylem and occasional ectopic metaxylem, similar to a weak loss-of-function mp-mutant. Our results provide evidence that HD-ZIP III TFs directly affect auxin response and mediate a feed forward loop formed by MP and IAA20 that may focus and stabilize auxin response during vascular patterning and differentiation of xylem cell types.
Project description:The class III HD-ZIPtranscription factors regulate vascular patterning in Arabidopsis thaliana roots. In this expression study we compare the expression profile in root tips upon miR165 induction, after 6h, 10h and 24h. The results are presented in PHABULOSA mediates an auxin signaling loop to regulate vascular patterning in Arabidopsis by Christina Joy Müller, Ana Elisa Valdés, Guodong Wang, Prashanth Ramachandran, Lisa Beste, Daniel Uddenberg, and Annelie Carlsbecker, accepted for publication in Plant Physiology Nov. 2015. Plant vascular tissues, xylem and phloem, differentiate in distinct patterns from procambial cells as an integral transport system for water, sugars and signaling molecules. Procambium formation is promoted by high auxin levels activating class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs). In the root of Arabidopsis thaliana, HD-ZIP III TFs dose-dependently govern the patterning of the xylem axis, with higher levels promoting metaxylem cell identity in the central axis and lower levels protoxylem at its flanks. It is, however, unclear by what mechanisms the HD-ZIP III TFs control xylem axis patterning. Here we present data suggesting that an important mechanism is their ability to moderate auxin response. We found that changes in HD-ZIP III TF levels affect the expression of genes encoding core auxin response molecules. We show that one of the HD-ZIP III TFs, PHABULOSA, directly binds the promoter of both MONOPTEROS/AUXIN RESPONSE FACTOR5 (MP/ARF5), a key factor in vascular formation, and IAA20, encoding an AUX/IAA protein which is stable in the presence of auxin and able to interact with and repress MP activity. The double mutant of IAA20 and its closest homologue IAA30 forms ectopic protoxylem, while overexpression of IAA30 causes discontinuous protoxylem and occasional ectopic metaxylem, similar to a weak loss-of-function mp-mutant. Our results provide evidence that HD-ZIP III TFs directly affect auxin response and mediate a feed forward loop formed by MP and IAA20 that may focus and stabilize auxin response during vascular patterning and differentiation of xylem cell types.
Project description:ATHB8 is a member of HD-ZIP III family transcription factors and has a important role in vascular development. However, how ATHB8 regulates vascular development is still a mystery. Here, we identified specific genes downstream of ATHB8-GFP with transformed suspension culture cells in microarray experiments.