Regulation of abscisic acid concentration in leaves of field-grown pearl millet (Pennisetum americanum (L.) Leeke): the role of abscisic acid export

Diurnal changes in the ABA concn. in leaves of droughted, field-grown plants of P. americanum were not always correlated with changes in bulk leaf water potential. A rapid decline in ABA content of the leaves following its rise to a peak level in mid-morning, was observed in several time-course studies despite continued water stress. The possibility that the reduction in ABA in leaves was due to an elevated rate of its export was examined by measuring ABA concn. in developing panicles (possible sinks for leaf-produced ABA) and in leaves, and by comparing the amounts of ABA in ungirdled leaves and in leaves heat-girdled at the base of the lamina to block export. ABA concn. in panicles generally paralleled those in leaves, though the peak concn. of ABA in the morning in panicles occurred later than in the leaves in some samplings. Although girdling initially increased ABA concn., it did not prevent a subsequent fall which generally paralleled the decline observed in untreated leaves. The decrease in ABA that occurred despite the block to export and despite continuing stress was attributed to changes in the synthesis or metabolism of ABA within the leaf. The probable rate of export of ABA from leaves, calculated from the changes in its concn. due to girdling, was highest at the time of most rapid ABA accumulation and declined thereafter. The percentage export of recently assimilated C declined similarly. However, the probable absolute rate of export of photosynthate, computed from stomatal conductance and [14C]-export measurements, was not uniquely related to that of AB

Overexpression of a Common Wheat Gene TaSnRK2.8 Enhances Tolerance to Drought, Salt and Low Temperature in Arabidopsis

Drought, salinity and low temperatures are major factors limiting crop productivity and quality. Sucrose non-fermenting1-related protein kinase 2 (SnRK2) plays a key role in abiotic stress signaling in plants. In this study, TaSnRK2.8, a SnRK2 member in wheat, was cloned and its functions under multi-stress conditions were characterized. Subcellular localization showed the presence of TaSnRK2.8 in the cell membrane, cytoplasm and nucleus. Expression pattern analyses in wheat revealed that TaSnRK2.8 was involved in response to PEG, NaCl and cold stresses, and possibly participates in ABA-dependent signal transduction pathways. To investigate its role under various environmental stresses, TaSnRK2.8 was transferred to Arabidopsis under control of the CaMV-35S promoter. Overexpression of TaSnRK2.8 resulted in enhanced tolerance to drought, salt and cold stresses, further confirmed by longer primary roots and various physiological characteristics, including higher relative water content, strengthened cell membrane stability, significantly lower osmotic potential, more chlorophyll content, and enhanced PSII activity. Meanwhile, TaSnRK2.8 plants had significantly lower total soluble sugar levels under normal growing conditions, suggesting that TaSnRK2.8 might be involved in carbohydrate metabolism. Moreover, the transcript levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI3, ABI4, ABI5), stress-responsive genes, including two ABA-dependent genes (RD20A, RD29B) and three ABA-independent genes (CBF1, CBF2, CBF3), were generally higher in TaSnRK2.8 plants than in WT/GFP controls under normal/stress conditions. Our results suggest that TaSnRK2.8 may act as a regulatory factor involved in a multiple stress response pathways

Energetics of Displacing Water Molecules from Protein Binding Sites: Consequences for Ligand Optimization

A strategy in drug design is to consider enhancing the affinity of lead molecules with structural modifications that displace water molecules from a protein binding site. Because success of the approach is uncertain, clarification of the associated energetics was sought in cases where similar structural modifications yield qualitatively different outcomes. Specifically, free energy perturbation calculations were carried out in the context of Monte Carlo statistical mechanics simulations to investigate ligand series that feature displacement of ordered water molecules in the binding sites of scytalone dehydratase, p38-αMAP kinase, and EGFR kinase. The change in affinity for a ligand modification is found to correlate with the ease of displacement of the ordered water molecule. However, as in the EGFR example, the binding affinity may diminish if the free energy increase due to the removal of the bound water molecule is not more than compensated by the additional interactions of the water-displacing moiety. For accurate computation of the effects of ligand modifications, a complete thermodynamic analysis is shown to be needed. It requires identification of the location of water molecules in the protein-ligand interface and evaluation of the free energy changes associated with their removal and with the introduction of the ligand modification. Direct modification of the ligand in free-energy calculations is likely to trap the ordered molecule and provide misleading guidance for lead optimization