Analysis of targeted CYCD7;1 expression in seed development

D-type cyclins in plants are represented by seven conserved subgroups and play a major role in controlling cell division. Relatively little is understood of their role during seed development, although their expression pattern has been characterized and ectopic expression of CYCD3;1 has previously been shown to disrupt normal embryo development. Here the consequences of ectopic expression of CYCD7;1 using the early endosperm-specific promoter FWA in developing Arabidopsis seeds were investigated. Ectopic CYCD7;1 expression in the maternal central cell prior to fertilization, and in the endosperm from fertilization until cellularization resulted in seeds up to 45% larger. Seed enlargement was accompanied seed lethality, shown to be due to a defect of development during early and mid stages of seed development. As expected from the maternal specific expression of the imprinted FWA promoter, seed size and lethality was dependent on maternal origin of the transgene. Larger seed size was correlated to mature embryo and seed coat outgrowth, and was due to cell proliferation rather than cell elongation. In particular, embryo development was accelerated during the early stages, suggesting these may be dependent on cell division rate, whereas later stages progressed at the same rate as WT seeds. Seed-targeted CYCD7;1 expression phenocopies (1) the nucleus proliferation in the endosperm prior to fertilization observed in rbr and fis-class mutants and (2) the seed enlargement observed in paternal genome excess interploidy crosses. These suggest that CYCD7;1 may act through the RBR pathway to promote cell proliferation and modify imprinting in the endosperm, thereby influencing the parental genome balance. Mechanistically, CYCD7;1 did not interact directly with CDKA;1 but the interaction was promoted in presence of the inhibitor of CDK, ICK1/KRP1 or ICK2/KRP2 in a yeast-three-hybrid assay. However, loss of either KRP1 or KRP2 in respective mutant backgrounds did not prevent the seed enlargement phenotype

Seed size plasticity in response to embryonic lethality conferred by ectopic CYD activation is dependent on plant architecture

The size of seeds is the result of cell proliferation and growth in the three seed compartments: the embryo, endosperm and integuments. Targeting expression of the D-type cyclin CYCD7;1 to the central cell and early endosperm (FWA:CYCD7;1) triggered nuclear divisions and partial ovule abortion, reducing seed number in each silique and leading to increased seed size. A similar effect on seed size was observed with other segregating embryo lethal mutations, suggesting caution is needed in interpreting apparent seed size phenotypes. Here, we show that the positive effect of FWA:CYCD7;1 on Arabidopsis seed size is modulated by the architecture of the mother plant. Larger seeds were produced in FWA:CYCD7;1 lines with unmodified inflorescences, and also upon removal of side branches and axillary stems. This phenotype was absent from inflorescences with increased axillary floral stems produced by pruning of the main stem. Given this apparent confounding influence of resource allocation on transgenes effect, we conclude that plant architecture is a further important factor to consider in appraising seed phenotypes

Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis

All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more complete digestion of chromatin to nucleosomes. We conclude that wide-spectrum analysis of the Arabidopsis genome by differential MNase digestion allows detection of sensitive features hereto obscured, and the comparisons between genome-wide subNSP profiles reveals dynamic changes in their distribution, particularly at distinct genomic locations (i.e. 5’UTRs). The method here employed allows insight into the complex influence of genetic and extrinsic factors in modifying the sub-nucleosomal landscape in association with transcriptional changes

WOX5 suppresses CYCLIN D activity to establish quiescence at the center of the root stem cell niche

In Arabidopsis, stem cells maintain the provision of new cells for root growth. They surround a group of slowly dividing cells named the quiescent center (QC), and, together, they form the stem cell niche (SCN). The QC acts as the signaling center of the SCN, repressing differentiation of the surrounding stem cells [ 1] and providing a pool of cells able to replace damaged stem cells [ 2 and 3]. Maintenance of the stem cells depends on the transcription factor WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the QC [ 4]. However, the molecular mechanisms by which WOX5 promotes stem cell fate and whether WOX5 regulates proliferation of the QC are unknown. Here, we reveal a new role for WOX5 in restraining cell division in the cells of the QC, thereby establishing quiescence. In contrast, WOX5 and CYCD3;3/CYCD1;1 both promote cell proliferation in the nascent columella. The additional QC divisions occurring in wox5 mutants are suppressed in mutant combinations with the D type cyclins CYCD3;3 and CYCD1;1. Moreover, ectopic expression of CYCD3;3 in the QC is sufficient to induce cell division in the QC. WOX5 thus suppresses QC divisions that are otherwise promoted by CYCD3;3 and CYCD1;1, in part by interacting with the CYCD3;3 promoter to repress CYCD3;3 expression in the QC. Therefore, we propose a specific role for WOX5 in initiating and maintaining quiescence of the QC by excluding CYCD activity from the QC

Phytotracker, an information management system for easy recording and tracking of plants, seeds and plasmids

Background A large number of different plant lines are produced and maintained in a typical plant research laboratory, both as seed stocks and in active growth. These collections need careful and consistent management to track and maintain them properly, and this is a particularly pressing issue in laboratories undertaking research involving genetic manipulation due to regulatory requirements. Researchers and PIs need to access these data and collections, and therefore an easy-to-use plant-oriented laboratory information management system that implements, maintains and displays the information in a simple and visual format would be of great help in both the daily work in the lab and in ensuring regulatory compliance. Results Here, we introduce ‘Phytotracker’, a laboratory management system designed specifically to organise and track plasmids, seeds and growing plants that can be used in mixed platform environments. Phytotracker is designed with simplicity of user operation and ease of installation and management as the major factor, whilst providing tracking tools that cover the full range of activities in molecular genetics labs. It utilises the cross-platform Filemaker relational database, which allows it to be run as a stand-alone or as a server-based networked solution available across all workstations in a lab that can be internet accessible if desired. It can also be readily modified or customised further. Phytotracker provides cataloguing and search functions for plasmids, seed batches, seed stocks and plants growing in pots or trays, and allows tracking of each plant from seed sowing, through harvest to the new seed batch and can print appropriate labels at each stage. The system enters seed information as it is transferred from the previous harvest data, and allows both selfing and hybridization (crossing) to be defined and tracked. Transgenic lines can be linked to their plasmid DNA source. This ease of use and flexibility helps users to reduce their time needed to organise their plants, seeds and plasmids and to maintain laboratory continuity involving multiple workers. Conclusion We have developed and used Phytotracker for over five years and have found it has been an intuitive, powerful and flexible research tool in organising our plasmid, seed and plant collections requiring minimal maintenance and training for users. It has been developed in an Arabidopsis molecular genetics environment, but can be readily adapted for almost any plant laboratory research

AINTEGUMENTA and the D-type cyclin CYCD3;1 independently contribute to petal size control in Arabidopsis: evidence for organ size compensation being an emergent rather than a determined property

Plant lateral aerial organ (LAO) growth is determined by the number and size of cells comprising the organ. Genetic alteration of one parameter is often accompanied by changes in the other, such that the overall effect on final LAO size is minimized, suggested to be caused by an active organ level ‘compensation mechanism’. For example, the aintegumenta (ant) mutant exhibits reduced cell number but increased cell size in LAOs. The ANT transcription factor regulates the duration of the cell division phase of LAO growth, and its ectopic expression is correlated with increased levels of the cell cycle regulator CYCD3;1. This has previously led to the suggestion that ANT regulates CYCD3;1. It is shown here that while ANT is required for normal cell proliferation in petals, CYCD3;1 is not, suggesting that ANT does not regulate CYCD3;1 during petal growth. Moreover CYCD3;1 expression was similar in wild-type and ant-9 flowers. In contrast to the compensatory changes between cell size and number in ant mutants, cycd3;1 mutants show increased petal cell size unaccompanied by changes in cell number, leading to larger organs. However, loss of CYCD3;1 in the ant-9 mutant background leads to a phenotype consistent with compensation mechanisms. These apparently arbitrary examples of compensation are reconciled through a model of LAO growth in which distinct phases of division and cell expansion occupy differing lengths of a defined overall growth window. This leads to the proposal that many observations of ‘compensation mechanisms’ might alternatively be more simply explained as emergent properties of LAO development

AINTEGUMENTA and the D-type cyclin CYCD3;1 independently contribute to petal size control in Arabidopsis: evidence for organ size compensation being an emergent rather than a determined property

Plant lateral aerial organ (LAO) growth is determined by the number and size of cells comprising the organ. Genetic alteration of one parameter is often accompanied by changes in the other, such that the overall effect on final LAO size is minimized, suggested to be caused by an active organ level ‘compensation mechanism’. For example, the aintegumenta (ant) mutant exhibits reduced cell number but increased cell size in LAOs. The ANT transcription factor regulates the duration of the cell division phase of LAO growth, and its ectopic expression is correlated with increased levels of the cell cycle regulator CYCD3;1. This has previously led to the suggestion that ANT regulates CYCD3;1. It is shown here that while ANT is required for normal cell proliferation in petals, CYCD3;1 is not, suggesting that ANT does not regulate CYCD3;1 during petal growth. Moreover CYCD3;1 expression was similar in wild-type and ant-9 flowers. In contrast to the compensatory changes between cell size and number in ant mutants, cycd3;1 mutants show increased petal cell size unaccompanied by changes in cell number, leading to larger organs. However, loss of CYCD3;1 in the ant-9 mutant background leads to a phenotype consistent with compensation mechanisms. These apparently arbitrary examples of compensation are reconciled through a model of LAO growth in which distinct phases of division and cell expansion occupy differing lengths of a defined overall growth window. This leads to the proposal that many observations of ‘compensation mechanisms’ might alternatively be more simply explained as emergent properties of LAO development

SubNSP recruitment occurs preferentially at active genes.

<p>Total coverage mapping of all sized DNA-binding particles for low-digested samples (Light-L1/L2 and Dark-L1/L2) demonstrates recruitment of subNSPs to specific TSS and UTR positions in the key light-response genes (A) Photosystem Subunit II (NPQ4, AT1G44575) and (B) Rubisco Small Subunit 3B (RBCS3B, AT5G38410). Observed subNSP recruitment is absent from dark-grown samples which are almost totally inactive (PSII—FC: -10.8, FDR = 2.59E-136, RBCS3B –FC: -9.46, FDR = 3.01E-076). Colour scale represents abundance of mapped fragments normalised for sequencing depth.</p

SubNSP organisation at transcription factor binding sites.

<p>TFBS positions were assayed for subNSP binding for light-responsive Transcription Factors (A) PIF3 (n = 1,930), (B) PIF4 (n = 20,252) and (C) the CCA1 (N = 59,249). Direct visualisation highlights the differential binding of the subNSP across the genome between growth conditions and identifying the TF response to the extrinsic irradiance changes.</p