Molecular Bases for Circadian Clocks

The r2r3-myb gene family in arabidopsis thaliana

Flowering is often triggered by exposing plants to appropriate day lengths. This response requires an endogenous timer called the circadian clock to measure the duration of the day or night. This timer also controls daily rhythms in gene expression and behavioural patterns such as leaf movements. Several Arabidopsis mutations affect both circadian processes and flowering time; but how the effect of these mutations on the circadian clock is related to their influence on flowering remains unknown. Here we show that expression of CONSTANS (CO), a gene that accelerates flowering in response to long days, is modulated by the circadian clock and day length. Expression of a CO target gene, called FLOWERING LOCUS T (FT), is restricted to a similar time of day as expression of CO. Three mutations that affect circadian rhythms and flowering time alter CO and FT expression in ways that are consistent with their effects on flowering. In addition, the late flowering phenotype of such mutants is corrected by overexpressing CO. Thus, CO acts between the circadian clock and the control of flowering, suggesting mechanisms by which day length regulates flowering time.

CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis

Circadian clocks are believed to confer an advantage to plants, but the nature of that advantage has been unknown. We show that a substantial photosynthetic advantage is conferred by correct matching of the circadian clock period with that of the external light-dark cycle. In wild type and in long- and short-circadian period mutants of Arabidopsis thaliana, plants with a clock period matched to the environment contain more chlorophyll, fix more carbon, grow faster, and survive better than plants with circadian periods differing from their environment. This explains why plants gain advantage from circadian control.

http://science.sciencemag.org/content/sci/309/5734/630.full.pdf

Plant Circadian Clocks Increase Photosynthesis, Growth, Survival, and Competitive Advantage

The circadian clock is a widespread cellular mechanism that underlies diverse rhythmic functions in organisms from bacteria and fungi, to plants and animals. Intense genetic analysis during recent years has uncovered many of the components and molecular mechanisms comprising these clocks. Although autoregulatory genetic networks are a consistent feature in the design of all clocks, the weight of evidence favours their independent evolutionary origins in different kingdoms.

Time zones: a comparative genetics of circadian clocks.

We have isolated the gene for a protein designated CCA1. This protein can bind to a region of the promoter of an Arabidopsis light-harvesting chlorophyll a/b protein gene, Lhcb1*3, which is necessary for its regulation by phytochrome. The CCA1 protein interacted with two imperfect repeats in the Lhcb1*3 promoter, AAA/cAATCT, a sequence that is conserved in Lhcb genes. A region near the N terminus of CCA1, which has some homology to the repeated sequence found in the DNA binding domain of Myb proteins, is required for binding to the Lhcb1*3 promoter. Lines of transgenic Arabidopsis plants expressing antisense RNA for CCA1 showed reduced phytochrome induction of the endogenous Lhcb1*3 gene, whereas expression of another phytochrome-regulated gene, rbcS-1A, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, was not affected. Thus, the CCA1 protein acts as a specific activator of Lhcb1*3 transcription in response to brief red illumination. The expression of CCA1 RNA was itself transiently increased when etiolated seedlings were transferred to light. We conclude that the CCA1 protein is a key element in the functioning of the phytochrome signal transduction pathway leading to increased transcription of this Lhcb gene in Arabidopsis.

/pdf/a-myb-related-transcription-factor-is-involved-in-the-11381gfvwf.pdf

A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene.

We have previously characterized a protein from Arabidopsis thaliana, called CA-1, that bound to a specific region of the Lhcb1*3 promoter. This binding activity was of interest because the sequence to which it bound is included in a portion of the promoter that is sufficient for phytochrome regulation and because the activity was absent in photomorphogenic mutant det1 seedlings (L. Sun, R.A. Doxsee, E. Harel, E.M. Tobin [1993] Plant Cell 5: 109-121). We have now directly tested whether the nucleotide sequence to which CA-1 binds is required for regulation of the transcription of this gene by phytochrome. A mutation that abolished CA-1 binding in vitro was introduced into a 1.15-kb segment of the Lhcb1*3 promoter, and both the wild-type and mutant promoter fragments were fused to a uidA reporter gene and used to stably transform A. thaliana. Ten different homozygous lines were examined for phytochrome responsiveness for each of the two constructs by assaying beta-glucuronidase activity. The wild-type construct showed normal phytochrome responsiveness. The mutant construct showed no phytochrome response, and the overall level of beta-glucuronidase activity in etiolated seedlings was decreased by about 2 orders of magnitude. We did not detect a response to a B photoreceptor other than phytochrome itself for either the wild-type or mutant construct. We conclude that information essential for both a high level of expression and phytochrome responsiveness is contained in a 27-bp region to which the CA-1 activity binds.

A Region of the Arabidopsis Lhcb1*3 Promoter That Binds to CA-1 Activity Is Essential for High Expression and Phytochrome Regulation

One approach towards understanding the transduction pathways of phytochromes is the selection of mutants impaired in various steps. We report here the construction of an inducible counter-selection system for such mutants employing the enzyme nitrate reductase. This enzyme can convert the benign substrate analogue chlorate to the toxic product chlorite, resulting in severe growth inhibition. An Arabidopsis thaliana nitrate reductase gene (Nial*2) was placed under the regulation of an Arabidopsis thaliana light-harvesting chlorophyll a/b protein (Lhcb1*3) promoter that is phytochrome-responsive. The chimeric Lhcb::Nia gene was transformed into A. thaliana. Homozygous transformant lines were selected and grown in the absence of nitrate and the presence of L-glutamine, conditions that substantially inhibited the expression of the endogenous nitrate reductase genes. In darkness seedlings of the transformed lines were resistant to chlorate; however, when seedlings were grown with intermittent red light, increased sensitivity to chlorate was observed. This sensitivity was correlated with an increase in both Nia1*2 RNA levels and nitrate reductase activity. The resistant seedlings were clearly distinguishable from the sensitive ones based on hypocotyl length, with no overlap in this parameter between the two populations. Thus, this system should allow for the selection of mutants that are impaired in phytochrome regulation of the transcription of Lhcb genes.

A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway.

Hydroponics is a farming technique for growing plants with mineral nutrients using a soil-free medium. The plant roots are submerged in soil-free media, such as vermiculite or perlite, or just in mineral nutrient solutions. This allows for high production yields throughout the year with less water and agro-chemical inputs. Consequently, hydroponics is considered a sustainable agriculture technology. Hydroponically grown crops are usually protected from the diseases transmitted through soil or animals in open fields. Therefore, they require fewer chemicals for pest control and are safer than conventionally grown crops in terms of possible chemical contamination. Nevertheless, hydroponics guarantees neither plant health nor the microbial safety of fresh produce. In the case of microbial contamination by human pathogens, unlike soil-grown crops, the pathogens may rapidly spread through the circulating water and simultaneously infect all the plants in the facility. This review summarizes the up-to-date knowledge regarding the microbial safety of hydroponically grown crops and discusses the role of the hydroponic system in reducing the microbial hazards for leafy and fruity crops as well as the potential risks for contamination by human pathogens. Finally, it outlines the approaches and the available science-based practices to ensure produce safety. The contamination risk in hydroponic systems may be diminished by using novel planting materials and the appropriate decontamination treatment of a recirculating liquid substrate; by modulating the microbiota interactions; and by following strict phytosanitary measures and workers’ hygienic practices. There is a timely need to adopt measures, such as the Good Agricultural Practice (GAP) guidelines, to mitigate the risks and ensure safe hydroponically grown vegetables for consumers.

/pdf/hydroponic-agriculture-and-microbial-safety-of-vegetables-1wsofp2b.pdf

Hydroponic Agriculture and Microbial Safety of Vegetables: Promises, Challenges, and Solutions

INTRODUCTION
Cannabis sativa L. inflorescences are rich in secondary metabolites, particularly cannabinoids. The most common techniques for elucidating cannabinoid composition are expensive technologies, such as high-pressure liquid chromatography (HPLC).


OBJECTIVES
We aimed to develop and evaluate the performance of a novel fluorescence spectroscopy-based method coupled with N-way partial least squares regression (N-PLS-R) and partial least squares discriminant analysis (PLS-DA) models to replace the expensive chromatographic methods for preharvest cannabinoid quantification.


METHODOLOGY
Fresh medicinal cannabis inflorescences were collected and ethanol extracts were prepared. Their excitation-emission spectra were measured using fluorescence spectroscopy and their cannabinoid contents were determined by HPLC-PDA. Subsequently, N-PLS-R and PLS-DA models were applied to the excitation-emission matrices (EEMs) for cannabinoid concentration prediction and cultivar classification, respectively.


RESULTS
The N-PLS-R model was based on a set of EEMs (n = 82) and provided good to excellent quantification of (-)-Δ9-trans-tetrahydrocannabinolic acid, cannabidiolic acid, cannabigerolic acid, cannabichromenic acid, and (-)-Δ9-trans-tetrahydrocannabinol (R2 CV and R2 pred  > 0.75; RPD > 2.3 and RPIQ > 3.5; RMSECV/RMSEC ratio < 1.4). The PLS-DA model enabled a clear distinction between the four major classes studied (sensitivity, specificity, and accuracy of the prediction sets were all ≥0.9).


CONCLUSIONS
The fluorescence spectral region (excitation 220-400 nm, emission 280-550 nm) harbors sufficient information for accurate prediction of cannabinoid contents and accurate classification using a relatively small data set.

Novel fluorescence spectroscopy method coupled with N-PLS-R and PLS-DA models for the quantification of cannabinoids and the classification of cannabis cultivars.

David Kenigsbuch

Papers

A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene.

A Region of the Arabidopsis Lhcb1*3 Promoter That Binds to CA-1 Activity Is Essential for High Expression and Phytochrome Regulation

A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway.

Hydroponic Agriculture and Microbial Safety of Vegetables: Promises, Challenges, and Solutions

Novel fluorescence spectroscopy method coupled with N-PLS-R and PLS-DA models for the quantification of cannabinoids and the classification of cannabis cultivars.