This study developed an approach for remote estimation of Vegetation Fraction (VF) and Flower Fraction (FF) in oilseed rape, which is a crop species with conspicuous flowers during reproduction. Canopy reflectance in green, red, red edge and NIR bands was obtained by a camera system mounted on an unmanned aerial vehicle (UAV) when oilseed rape was in the vegetative growth and flowering stage. The relationship of several widely-used Vegetation Indices (VI) vs. VF was tested and found to be different in different phenology stages. At the same VF when oilseed rape was flowering, canopy reflectance increased in all bands, and the tested VI decreased. Therefore, two algorithms to estimate VF were calibrated respectively, one for samples during vegetative growth and the other for samples during flowering stage. The results showed that the Visible Atmospherically Resistant Index (VARI_green) worked most accurately for estimating VF in flower-free samples with an Root Mean Square Error (RMSE) of 3.56%, while the Enhanced Vegetation Index (EVI2) was the best in flower-containing samples with an RMSE of 5.65%. Based on reflectance in green and NIR bands, a technique was developed to identify whether a sample contained flowers and then to choose automatically the appropriate algorithm for its VF estimation. During the flowering season, we also explored the potential of using canopy reflectance or VIs to estimate FF in oilseed rape. No significant correlation was observed between VI and FF when soil was visible in the sensor’s field of view. Reflectance at 550 nm worked well for FF estimation with coefficient of determination (R²) above 0.6. Our model was validated in oilseed rape planted under different nitrogen fertilization applications and in different phenology stages. The results showed that it was able to predict VF and FF accurately in oilseed rape with RMSE below 6%.

Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states—a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pK_a value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pH-sensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals.

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function.

The interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13^NTD) and Ub monomer has been characterized, while it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13^NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have determined the complex structure between Rpn13^NTD and K48-diUb. In this structure, Rpn13^NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13^NTD similarly as the Ub monomer in the known Rpn13^NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13^NTD. Thus, a charge-reversal mutation in Rpn13^NTD weakens the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, physical blockage of the access of the distal Ub to Rpn13^NTD with a proximity-attached Ub monomer can disrupt the interaction between Rpn13 and K48-diUb. Taken together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.

Chlorophyll fluorescence (ChlF) is an important signature of photosynthesis to evaluate plant response to the environment. We explored an approach to estimate an important leaf ChlF-derived parameter, the intrinsic efficiency of photosystem II photochemistry ($Fv/Fm$), using spectral indices calculated from leaf reflectance measured by a hyperspectral radiometer. It is observed that leaf chlorophyll content closely related to $Fv/Fm$ in nonstressed leaves, thus the indices developed for chlorophyll estimation were successfully used to estimate $Fv/Fm$. For leaves under short-term stress, $Fv/Fm$ dropped dramatically while leaf chlorophyll content remained almost the same. Compared to leaf chlorophyll content, reflectance was more sensitive to $Fv/Fm$ variations. As $Fv/Fm$ decreased, the slope of reflectance in the spectrum range of 700 to 900 nm obviously increased, and the first derivative reflectance in the red edge and infrared (NIR) regions was highly correlated with $Fv/Fm$. The indices using longwave red edge and NIR reflectance ($NDRE740$ and $CI740$) worked well for $Fv/Fm$ retrieval in both stressed and nonstressed leaves with the coefficients of determination ($R2$) above 0.72 and normalized root-mean-square errors below 0.16. Note that the relationships $NDRE740$ and $CI740$ versus $Fv/Fm$ were significantly different between nonstressed and stressed leaves, which may give a good implication to detect short-term stress occurrence.