Activation of embryogenic cell division in leaf protoplast-derived alfalfa cells: the role of auxin and stress

Leaf protoplast-derive d cells of the embryogenic alfalfa genotype (Medicago sativa ssp. varia A2) follow different fate if cultured in the presence of 0.25, 1 or 10 uM 2,4 - dichlorophenoxyacetic acid (2,4-D). Cells grow n in the presence of the highest auxin (2,4-D) concentration become embryogenic and can develop into somatic embryos if subcultured into fresh medium with the lower 2,4- D level. Cells cultured at the lower auxin concentrations from the beginning develop into elongated differentiated cells. In order to reveal physiological changes that characterize the reactivation of cell division in resting cells as well as the transition of somatic plant cells to an embryogenic state, morphological, cell division, intracellular pH and stress-related parameters have been determined during the first five days of parallel cultures at the above 2,4-D concentrations in combination with stress treatments

From Single Cell to Plants: Mesophyll Protoplasts as a Versatile System for Investigating Plant Cell Reprogramming

Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is an extreme example of such plasticity, whereby somatic cells have the potential to form plants via direct shoot organogenesis or somatic embryogenesis in response to various exogenous and/or endogenous signals. Protoplasts provide one of the most suitable systems for investigating molecular mechanisms of totipotency, because they are e ectively single cell populations. In this review, we consider the current state of knowledge of the mechanisms that induce cell proliferation from individual, di erentiated somatic plant cells. We highlight initial explant metabolic status, ploidy level and isolation procedure as determinants of successful cell reprogramming. We also discuss the importance of auxin signalling and its interaction with stress-regulated pathways in governing cell cycle induction and further stages of plant cell totipotency

Interplay of the two ancient metabolites auxin and MEcPP regulates adaptive growth.

The ancient morphoregulatory hormone auxin dynamically realigns dedicated cellular processes that shape plant growth under prevailing environmental conditions. However, the nature of the stress-responsive signal altering auxin homeostasis remains elusive. Here we establish that the evolutionarily conserved plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) controls adaptive growth by dual transcriptional and post-translational regulatory inputs that modulate auxin levels and distribution patterns in response to stress. We demonstrate that in vivo accumulation or exogenous application of MEcPP alters the expression of two auxin reporters, DR5:GFP and DII-VENUS, and reduces the abundance of the auxin-efflux carrier PIN-FORMED1 (PIN1) at the plasma membrane. However, pharmacological intervention with clathrin-mediated endocytosis blocks the PIN1 reduction. This study provides insight into the interplay between these two indispensable signaling metabolites by establishing the mode of MEcPP action in altering auxin homeostasis, and as such, positioning plastidial function as the primary driver of adaptive growth

Protocol: An improved and universal procedure for whole-mount immunolocalization in plants

Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment

ANALISA LAPORAN KEUANGAN SEBAGAI DASAR PENGUKURAN TINGKAT PROFITABILITAS PADA PERUSAHAAN DAERAH AIR MINUM (PDAM) KABUPATEN KARAWANG PERIODE 2002-2004

Perusahaan Daerah Air Minum (PDAM) Kabupaten Karawang adalah perusahaan yang bergerak dalam bidang jasa penyediaan air minum yang didistribusikan kepada masyarakat. Berdasarkan laporan keuangan PDAM Kabupaten Karawang yang diambil dalam penelitian pada periode 2002 hingga periode 2004 menggambarkan adanya fluktuasi pada salah satu rasio profitabilitasnya, yaitu pada rasio Basic Earning Power. Adapun tujuan yang ingin dicapai dalam penelitian ini adalah untuk mengetahui kondisi keuangan perusahaan, mengetahui analisa laporan keuangan sebagai dasar pengukuran profitabilitas perusahaan serta mengetahui faktor-faktor yang mempengaruhi laporan keuangan terhadap profitabilitas perusahaan. Metode penelitian yang digunakan adalah metode penelitian deskriptif dan teknik pengumpulan data yang digunakan adalah studi kepustakaan dan studi lapangan yang terdiri dari wawancara terstruktur dan observasi non partisipan. Penelitian tersebut dilakukan dengan menggunakan metode analisis data horizontal, sedangkan teknik analisis data yang dipergunakan adalah dengan teknik analisis trend dan analisis rasio profitabilitas. PDAM Kabupaten Karawang didalam perkembangan tingkat profitabilitasnya mengalami fluktuasi, terutama pada rasio basic earning powernya. Tingkat fluktuasi atas rasio profitabilitas perusahaan tersebut dipengaruhi oleh kenaikan tarif dasar air per m3 sebesar 40% hingga akhir periode 2004, selain itu didukung pula dengan peningkatan frekuensi sambungan rekening baru dengan mayoritas konsumen dari golongan niaga. Berdasarkan uraian diatas, maka hasil analisa laporan keuangan dapat dijadikan sebagai dasar pengukuran tingkat profitabilitas perusahaan. Faktor-faktor yang mempengaruhi laporan keuangan terhadap tingkat profitabilitas pada PDAM Karawang yaitu tingginya biaya pengolahan air melebihi biaya pembelian air baku, lemahnya kinerja bagian Satuan Pengendalian Internal sehingga memudahkan terjadinya penyimpangan disipliner serta sering terjadinya keterlambatan penyampaian laporan keuangan cabang sehingga penyusunan laporan pusat seringkali tidak tetap dan cenderung kadaluarsa. Saran yang peneliti kemukakan yaitu PDAM Karawang seharusnya menerapkan metode komputerisasi yang terkoneksi pada seluruh kantor cabang serta kerjasama dengan pihak luar (bank), guna memperkecil penyimpangan, mempermudah koordinasi serta memudahkan proses penyampaian data laporan keuangan setiap cabang sehingga diharapkan tidak akan terjadi lagi keterlambatan, yang kedua meningkatkan kinerja Satuan Pengendalian Internal agar mampu mengurangi bentuk penyimpangan

Methods of In Situ Quantitative Root Biology

When dealing with plant roots, a multiscale description of the functional root structure is needed. Since the beginning of 21st century, new devices such as laser confocal microscopes have been accessible for coarse root structure measurements, including three-dimensional (3D) reconstruction. Most researchers are familiar with using simple 2D geometry visualization that does not allow quantitative determination of key morphological features from an organ-like perspective. We provide here a detailed description of the quantitative methods available for 3D analysis of root features at single-cell resolution, including root asymmetry, lateral root analysis, cell size and nuclear organization, cell-cycle kinetics, and chromatin structure analysis. Quantitative maps of the root apical meristem (RAM) are shown for different species, including Arabidopsis thaliana (L.), Heynh, Nicotiana tabacum L., Medicago sativa L., and Setaria italica (L.) P. Beauv. The 3D analysis of the RAM in these species showed divergence in chromatin organization and cell volume distribution that might be used to study root zonation for each root tissue. Detailed protocols and possible pitfalls in the usage of the marker lines are discussed. Therefore, researchers who need to improve their quantitative root biology portfolio can use them as a reference