Effects of Leaf Excision and Sample Storage Methods on Spectral Reflectance by Foliage of Giant Reed, Arundo donax

Research was conducted to evaluate the effects of leaf excision and sample storage methods on spectral reflectance by foliage of giant reed, Arundo donax, an invasive weed which has caused extensive damage in many areas of the Rio Grande Basin in Texas and Mexico. Within 24 hours of excision, A. donax leaves exposed to ambient laboratory conditions (room temperature under natural lighting conditions) exhibited two trends indicative of physiological stress: 1) small but significant increases in reflectance of blue and red wavelengths (400-500 nm and 600-700 nm, respectively) and 2) a substantial reduction in reflectance of near-infrared (NIR) wavelengths (700-1,100 nm). A similar but less pronounced trend was evident among leaf samples held within conventional paper sacks. Leaf samples held within sealed plastic bags (Glad-Bags) under two types of lighting conditions (natural light and artificial darkness) and temperature regimes (room temperature vs artificially cooled) exhibited slight but significant increases in both visible and NIR wavelengths (a trend that was also evident in attached foliage), al-though no evidence of physiological stress was detected during a 96-hour observation period. These trends indicate that accurate spectral measurements may be obtained from samples of A. donax foliage under for periods up to 72 - 96 hours following excision if such samples are transported and maintained in suitable containers designed to minimize effects of desiccation

Techniques to Facilitate the Acquisition of Accurate Spectral Measurements and Multispectral Imagery of Plant Foliage Under Artificial Lighting Conditions

Spectral measurements obtained in the laboratory under artificial lighting sources have been used for many years to develop spectral ‘libraries’ for various soil types, rocks and minerals, and other inanimate features occurring on or near the earth’s surface. Quartz halogen lamps have been shown to emit all of the electromagnetic (EMR) wavelengths required for the acquisition of quality multispectral imagery, and various techniques have been developed to facilitate the acquisition of accurate spectral measurements using such artificial lighting sources. Our objectives in this study were to evaluate the several factors of critical importance in obtaining accurate spectral measurements for plant foliage in the laboratory, including the effects of leaf orientation, excision, and background reflectance on reflectance of visible and NIR wavelengths. Results demonstrated that accurate spectral measurements and imagery of various types of foliage may be obtained using quartz halogen lighting provided that excised leaf samples are placed on an NIR-absorbent background and samples are maintained in containers designed to minimize desiccation

Loss of Rb-E2F repression results in caspase-8-mediated apoptosis through inactivation of focal adhesion kinase

Molecular hardwiring of the cell cycle to the apoptotic machinery is a critical tumor suppressor mechanism for eliminating hyperproliferative cells. Deregulation of the Rb-E2F repressor complex by genetic deletion or functional inhibition of Rb triggers apoptosis through both the intrinsic (caspase-9 mediated) and extrinsic (caspase-8 mediated) death pathways. Induction of the intrinsic pathway has been studied extensively and involves release of free E2F and direct transcriptional activation of E2F-responsive apoptotic genes such as ARF, APAF1, and CASP9. In contrast, the mechanisms leading to activation of the extrinsic pathway are less well understood. There is growing evidence that Rb-E2F perturbation induces the extrinsic pathway, at least in part, through derepression (as opposed to transactivation) of apoptotic genes. Here, we explore this possibility using cells in which Rb-E2F complexes are displaced from promoters without stimulating E2F transactivation. This derepression of Rb-E2F-regulated genes leads to apoptosis through inactivation of focal adhesion kinase and activation of caspase-8. These findings reveal a new mechanistic link between Rb-E2F and the extrinsic (caspase 8-mediated) apoptotic pathway

Protein tyrosine phosphatases PTP-1B, SHP-2, and PTEN facilitate Rb/E2F-associated apoptotic signaling.

To maintain tissue homeostasis, apoptosis is functionally linked to the cell cycle through the retinoblastoma (Rb)/E2F pathway. When the Rb tumor suppressor protein is functionally inactivated, E2F1 elicits an apoptotic response through both intrinsic (caspase-9 mediated) and extrinsic (caspase-8 mediated) apoptotic pathways in order to eliminate hyperproliferative cells. Rb/E2F-associated apoptosis has been demonstrated to be associated with the loss of constitutive transcriptional repression by Rb/E2F complexes and mediated by caspase-8. Protein tyrosine phosphatases (PTPs) PTP-1B and SHP-2 have been previously shown to be directly activated by loss of Rb/E2F repression during Rb/E2F-associated apoptosis. In this current study, we demonstrate that the PTEN tumor suppressor is also directly activated by loss of Rb/E2F repression. We also demonstrate that PTP-1B, SHP-2, and PTEN play a functional role in Rb/E2F-associated apoptosis. Knockdown of PTP1B, SHP2, or PTEN expression with small interfering RNA (siRNA) in apoptotic cells increases cell viability and rescues cells from the Rb/E2F-associated apoptotic response. Furthermore, rescue from apoptosis coincides with inhibition of caspase-8 and caspase-3 cleavage (activation). Our results indicate PTP-1B, SHP-2, and PTEN all play a functional role in Rb/E2F-associated apoptotic signal transduction and provide further evidence that PTP-1B, SHP-2, and PTEN can contribute to tumor suppression through an Rb/E2F-associated mechanism

<i>PTEN</i> transcription is directly regulated by Rb/E2F complexes.

<p>ER-dnE2F1 cells were treated IPTG and with 4-OHT to induce Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Total RNA was isolated at the indicated time points following induction of apoptosis and used for qPCR analysis for two independent experiments performed in triplicate. Results are expressed as the mean + standard deviation. (B) Chromatin was isolated from the cells, sheared and immunoprecipitated with control mouse IgG or E2F1 antibody. A ∼300 bp sequence within the PTEN promoter region was amplified by PCR. GAPDH was used as control. (−) Untreated; (+) Treated; (NA) No antibody negative control; (*) No chromatin negative control.</p

SHP-2 and PTEN protein expression increases during Rb/E2F-associated apoptosis.

<p>ER-dnE2F1 cells were treated with 4-OHT for the indicated times to induce Rb/E2F-associated apoptosis. Control cells (0 h) were left untreated. (A) Total protein was isolated from lysated cells at the indicated time points following induction of apoptosis, resolved by SDS-PAGE, and immunoblotted with antibodies specific for PTP-1B, SHP-2, PTEN and β-actin. (B) Relative expression levels of PTP-1B, SHP-2, and PTEN were quantified by densitometry. (h) Hours.</p

Loss of PTP expression in apoptotic cells leads to an increase in cell proliferation.

<p>PTP gene expression was silenced in ER-dnE2F1 cells with siRNA specific for PTP-1B (+PTP-1B), SHP-2 (+SHP-2), or PTEN (+PTEN). Uninduced and induced control cells were transfected with scrambled siRNA. Then cells were treated with 4-OHT for 48 h to initiate Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Total protein was isolated from lysated cells, resolved by SDS-PAGE, and immunoblotted with antibodies specific for PTP-1B, SHP-2, PTEN and β-actin. (B) Morphological changes in uninduced and induced ER-dnE2F1 cells were compared with morphological changes in induced cells after knockdown of PTPs. (C) The number of cells within three random fields (100X) were counted and results are expressed as the mean + standard deviation. (D) Cell viability was determined by WST-8 assay in two independent experiments performed in triplicate. Results are expressed as the mean + standard deviation. (*) <i>p</i><0.05; (**) <i>p</i><0.01; (***) <i>p</i><0.001.</p

Knockdown of PTP expression leads to a reduction in caspase activity.

<p>ER-dnE2F1 cells were transfected with siRNA specific for PTP-1B (+PTP-1B), SHP-2 (+SHP-2), or PTEN (+PTEN). Uninduced and induced control cells were transfected with scrambled siRNA. Then cells were treated with 4-OHT for 24 or 48 h to initiate Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Caspase-3/CPP32 cleavage was measured by colorimetric assay in three independent experiments. Results are expressed as the mean + standard error. (B) FLICE/Caspase-8 cleavage was measured by colorimetric assay in three independent experiments. Results are expressed as the mean + standard error. (C) Total protein was isolated from lysated cells at 24 h following induction of apoptosis, resolved by SDS-PAGE, and immunoblotted with antibodies specific for cleaved caspase-3 (Denoted by arrow), caspase-3 and β-actin. (*) <i>p</i><0.05; (**) <i>p</i> = 0.01; (***) <i>p</i><0.01.</p