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Early Leaf Development: A not so gradual process

ABSTRACT: Early leaf growth is sustained by cell proliferation and subsequent cell expansion that initiates at the leaf tip and proceeds in a basipetal direction. Using detailed kinematic and gene expression studies to map these stages during early development of the third leaf of Arabidopsis thaliana we showed that the cell cycle arrest front did not progress gradually down the leaf, but rather was established and abolished abruptly. Interestingly, leaf greening and stomatal patterning followed a similar basipetal pattern, but proliferative pavement cell and formative meristemoid divisions were uncoordinated in respect to onset and persistence. Genes differentially expressed during the transition from cell proliferation to expansion were enriched in genes involved in cell cycle, photosynthesis, and chloroplast retrograde signaling. Proliferating primordia treated with norflurazon, a chemical inhibitor of retrograde signaling, showed inhibited onset of cell expansion. Hence, differentiation of the photosynthetic machinery is important for regulating the exit from proliferation. GSM839070-GSM839081: Total RNA was extracted using RNeasy plant mini kit (Qiagen) from leaf 3 of Arabidopsis seedlings grown in vitro and treated or not treated with norflurazon (NF) at 8 days after stratification (DAS) and harvested 24 and 48 hours after treatment. RNA from three biological repeats of each timepoint and treatment were submitted to AGRONOMICS1 array hybridization. GSM839082-GSM839099: Total RNAs were extracted using RNeasy plant mini kit (Qiagen) from leaf 3 of Arabidopsis seedlings 8, 9, 10, 11, 12, and 13 days after stratification (DAS) and grown in vitro. RNAs from three biological repeats of each timepoint were submitted to AGRONOMICS1 array hybridization.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Megan Andriankaja

PROVIDER: E-GEOD-33936 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Leaf growth is a complex developmental process that is continuously fine-tuned by the environment. Various abiotic stresses, including mild drought stress, have been shown to inhibit leaf growth in Arabidopsis thaliana (Arabidopsis), but the underlying mechanisms remain largely unknown. Here we identify the redundant Arabidopsis transcription factors ETHYLENE RESPONSE FACTOR 5 (ERF5) and ERF6 as master regulators which adapt leaf growth to environmental changes. ERF5 and ERF6 gene expression is induced very rapidly and specifically in actively growing leaves after sudden exposure to osmotic stress that mimics mild drought. Subsequently, enhanced ERF6 expression inhibits cell proliferation and leaf growth by a process involving GA and DELLA signaling. Using an ERF6 inducible overexpression line, we demonstrate that the GA-degrading enzyme GA2-OX6 is transcriptionally induced by ERF6 and that consequently DELLA proteins are stabilized. As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while growth of erf5erf6 loss-of-function mutants is less affected by stress. Next to its role in plant growth under stress, ERF6 also activates the expression of a plethora of osmotic stress-responsive genes, including the well-known stress tolerance genes STZ, MYB51 and WRKY33. Interestingly, the activation of the stress tolerance genes by ERF6 occurs independently from the ERF6-mediated growth inhibition. Together, these data fit into a leaf growth regulatory model in which ERF5 and ERF6 form a missing link between the previously observed stress-induced 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation and DELLA-mediated cell cycle exit and execute a dual role by regulating both stress tolerance and growth-inhibition. Samples were obtained from three independent experiments and from multiple plates within the experiment. Whole seedlings were harvested rapidly in an excess of RNAlaterÂ® solution (Ambion), and after overnight storage at 4Â°C, dissected under a binocular microscope on a cooling plate with precision microscissors. Dissected leaves were transferred to a new tube, frozen in liquid nitrogen, and ground with a Retsch machine and 3-mm metal balls. RNA was extracted with TriZol (Invitrogen) and further purified with the RNeasy Mini Kit (Qiagen). DNA digestion was done on columns with RNase-free DNase I (Roche). For the identification of genome-wide expression changes, samples of the strong ERF6-overexpressing line (ERF6IOE-S) and the control line (GFP:IOE) were harvested 4Â h after transfer to DEX. Two Âµg of pure RNA samples were hybridized to AGRONOMICS1 Arabidopsis Tiling Arrays (Rehrauer et al., 2010) at the VIB Microarray Facility (Leuven, Belgium).

Project description:Leaf senescence is an essential developmental process that involves altered regulation of thousands of genes and changes in many metabolic and signaling pathways resulting in massive physiological and structural changes in the leaf. The regulation of senescence is complex and although several senescence regulatory genes have been identified and characterized there is little information on how these individual regulators function globally in the control of the process. In this paper we use microarray analysis to obtain a high-resolution time course profile of gene expression during development of a single leaf over a three week period from just before full expansion to senescence. The multiple time points enable the use of highly informative clustering tools to reveal distinct time points at which signaling and metabolic pathways change during senescence. Analysis of motif enrichment in co-regulated gene clusters identifies clear groups of transcription factors active at different stages of leaf development and senescence. A novel experimental design strategy (A Mead et al, in preparation), based on the principle of the “loop design”, was developed to enable efficient extraction of information about key sample comparisons using a two-colour hybridisation experimental system. With 88 distinct samples (four biological replicates at each of 22 time points) to be compared, the experimental design included 176 two-colour microarray slides, allowing four technical replicates of each sample to be observed. Half of the slides were devoted to assessment of changes in gene expression between time points, using a simple loop design to link 11 samples from either the 7h time points or the 14h time points across the 11 sampling days, directly comparing samples collected on adjacent sampling days (i.e. 19 DAS with 21 DAS, 27 DAS with 29 DAS, etc.), and directly comparing the samples collected at 39 DAS with those collected at 19 DAS. Four separate loops were constructed for the 7h time points and for the 14h time points, using the arbitrary biological replicate labelling to identify the samples to be included in each loop. The remaining slides provided assessment of differences between the 7h and 14h samples and between the arbitrarily labelled biological replicates, with some further assessment of changes between sampling days. All direct comparisons (pairs of samples hybridised together on a slide) were between 7h and 14h samples collected on adjacent sampling days (i.e. 19 DAS with 21 DAS, etc.), including comparisons between samples collected at 39 DAS and at 19 DAS, and different arbitrarily labelled biological replicates. These 88 comparisons formed a single loop connecting all 88 treatments, therefore ensuring that the design was fully connected (allowing each sample to be compared with every other sample).

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Early Leaf Development: A not so gradual process

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