Functional Characterization of the GATA Transcription Factors GNC and CGA1 Reveals Their Key Role in Chloroplast Development, Growth, and Division in Arabidopsis

Chloroplasts develop from proplastids in a process that requires the interplay of nuclear and chloroplast genomes, but key steps in this developmental process have yet to be elucidated. Here, we show that the nucleus-localized transcription factors GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA1 (CGA1) regulate chloroplast development, growth, and division in Arabidopsis (Arabidopsis thaliana). GNC and CGA1 are highly expressed in green tissues, and the phytohormone cytokinin regulates their expression. A gnc cga1 mutant exhibits a reduction in overall chlorophyll levels as well as in chloroplast size in the hypocotyl. Ectopic overexpression of either GNC or CGA1 promotes chloroplast biogenesis in hypocotyl cortex and root pericycle cells, based on increases in the number and size of the chloroplasts, and also results in expanded zones of chloroplast production into the epidermis of hypocotyls and cotyledons and into the cortex of roots. Ectopic overexpression also promotes the development of etioplasts from proplastids in dark-grown seedlings, subsequently enhancing the deetiolation process. Inducible expression of GNC demonstrates that GNC-mediated chloroplast biogenesis can be regulated postembryonically, notably so for chloroplast production in cotyledon epidermal cells. Analysis of the gnc cga1 loss-of-function and overexpression lines supports a role for these transcription factors in regulating the effects of cytokinin on chloroplast division. These data support a model in which GNC and CGA1 serve as two of the master transcriptional regulators of chloroplast biogenesis, acting downstream of cytokinin and mediating the development of chloroplasts from proplastids and enhancing chloroplast growth and division in specific tissues

AirJump: Using Interfaces to Instantly Perform Simultaneous Extractions

Analyte isolation is an important process that spans a range of biomedical disciplines, including diagnostics, research, and forensics. While downstream analytical techniques have advanced in terms of both capability and throughput, analyte isolation technology has lagged behind, increasingly becoming the bottleneck in these processes. Thus, there exists a need for simple, fast, and easy to integrate analyte separation protocols to alleviate this bottleneck. Recently, a new class of technologies has emerged that leverages the movement of paramagnetic particle (PMP)-bound analytes through phase barriers to achieve a high efficiency separation in a single or a few steps. Specifically, the passage of a PMP/analyte aggregate through a phase interface (aqueous/air in this case) acts to efficiently “exclude” unbound (contaminant) material from PMP-bound analytes with higher efficiency than traditional washing-based solid-phase extraction (SPE) protocols (i.e., bind, wash several times, elute). Here, we describe for the first time a new type of “exclusion-based” sample preparation, which we term “AirJump”. Upon realizing that much of the contaminant carryover stems from interactions with the sample vessel surface (e.g., pipetting residue, wetting), we aim to eliminate the influence of that factor. Thus, AirJump isolates PMP-bound analyte by “jumping” analyte directly out of a free liquid/air interface. Through careful characterization, we have demonstrated the validity of AirJump isolation through comparison to traditional washing-based isolations. Additionally, we have confirmed the suitability of AirJump in three important independent biological isolations, including protein immunoprecipitation, viral RNA isolation, and cell culture gene expression analysis. Taken together, these data sets demonstrate that AirJump performs efficiently, with high analyte yield, high purity, no cross contamination, rapid time-to-isolation, and excellent reproducibility

Using Exclusion-Based Sample Preparation (ESP) to Reduce Viral Load Assay Cost.

Viral load (VL) measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD), accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1) and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP).71 patient samples with VLs ranging from 3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL) and high accuracy (average difference between methods of 0.08 log, R2 = 0.97). Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries

Loading and operation of ESP devices.

<p>A) An unfilled device with each well labeled. B) Aqueous reagents are first added to the ESP device. C) Oil is added last, between each aqueous reagent. D) Manually operated devices are held by ridges located around the periphery of the ESP device to prevent contact between the reagents and the operator’s hand. E) Optionally, PMPs can be mixed within each wash well to enhance purity. F) After operation, the eluent is removed from the ESP device via pipette.</p

Results of the ESP comparison trial.

<p>A) Comparison of RNA extraction between ESP and gold standard protocols; B) Comparison of low cost RT-qPCR reagents with gold standard reagents on ESP-extracted RNA; C) Breakdown of total assay costs for ESP / low cost reagent protocol.</p