Dynamics of soil penetration resistance, moisture depletion pattern and crop productivity determined by mechanized cultivation and lifesaving irrigation in zero till blackgram.

Rice fallow black gram is grown under the residual moisture situation as a relay crop in heavy texture montmorillonite clay soil under zero till condition. Since the crop is raised during post monsoon season, the crop often experiences terminal stress due to limited water availability and no rainfall. Surface irrigation in montmorillonite clay soil is determent to pulse crop as inundation causes wilting. Therefore, zero tilled rice fallow black gram has to be supplemented with micro irrigation at flowering stage (35 days after sowing) to alleviate moisture stress and to increase the productivity as well. Hence micro farm pond in a corner of one ha field was created to harvest the rain water during monsoon season and the same was utilized to supplement the crop with lifesaving irrigation through mobile sprinkler at flowering stage for the crop grown under conservation agriculture. Soil cracking is also the common phenomena of montmorillonite clay soil where evaporations losses would be more through crack surfaces. The present study was therefore conducted to study the changes in the soil physical properties, crop establishment and productivity in conjunction with mechanized sowing and harvest and supplemental mobile sprinkler irrigation. Sowing of black gram by broadcasting 10 days prior to the manual harvest of rice, manual drawn single row seed drill after the machine harvest of rice and sowing by broadcasting at 4 days prior to machine harvest of rice was experimented separately and in combination with lifesaving irrigation. Results indicated that the number of wheel passes and lifesaving irrigation had a very strong impact on soil penetration resistance and soil moisture. Combined harvester followed by no till seed drill increased the soil penetration resistance in all the layers (0-5 cm, 5-10 cm and 10-15 cm). Two passes of wheel increased the mean soil penetration resistance from 407 KPa to 502 KPa. The soil penetration resistance (0-5 cm) at harvest shown that black gram sown by manual broadcasting 10 days prior to manual harvest of paddy supplemented with life irrigation on 30 DAS reduced the soil penetration resistance from 690 Kpa to 500 Kpa, 740 Kpa to 600 Kpa and 760 Kpa to 620 Kpa respectively at 0-5 cm, 5-10 cm and 10-15 cm layer. In general, moisture depletion rate was rapid in the surface layer of 0-5 cm as compared to other layers of 5-10 cm and 10-15 cm up to 30 DAS (Flowering stage). The moisture content and the soil penetration resistance had an inverse relationship. The soil penetration resistance also had an inverse relationship with the root length in which the root length lowers as the soil penetration resistance increases. The soil crack measured at 60 DAS was deeper with no till seed drill (width of 3.94 cm and depth of 13.67 cm) which was mainly due to surface layer compaction. The relative water content, specific leaf weight and chlorophyll content were significantly improved through the supplemental irrigation given on 30 DAS irrespective of crop establishment methods. The results further indicated that compaction of ploughed layer in the moist soil due to combined harvester and no till seed drill had a negative impact on yield (457 kg ha-1), which was improved by 19.03 per cent due to increased soil moisture with supplemental irrigation. The mean yield increase across different treatments due to supplemental lifesaving irrigation through mobile sprinkler was 20.4 per cent.

Climate-based statistical regression models for crop yield forecasting of coffee in humid tropical Kerala, India.

A study on the variability of coffee yield of both Coffea arabica and Coffea canephora as influenced by climate parameters (rainfall (RF), maximum temperature (Tmax), minimum temperature (Tmin), and mean relative humidity (RH)) was undertaken at Regional Coffee Research Station, Chundale, Wayanad, Kerala State, India. The result on the coffee yield data of 30 years (1980 to 2009) revealed that the yield of coffee is fluctuating with the variations in climatic parameters. Among the species, productivity was higher for C. canephora coffee than C. arabica in most of the years. Maximum yield of C. canephora (2040 kg ha-1) was recorded in 2003-2004 and there was declining trend of yield noticed in the recent years. Similarly, the maximum yield of C. arabica (1745 kg ha-1) was recorded in 1988-1989 and decreased yield was noticed in the subsequent years till 1997-1998 due to year to year variability in climate. The highest correlation coefficient was found between the yield of C. arabica coffee and maximum temperature during January (0.7) and between C. arabica coffee yield and RH during July (0.4). Yield of C. canephora coffee had highest correlation with maximum temperature, RH and rainfall during February. Statistical regression model between selected climatic parameters and yield of C. arabica and C. canephora coffee was developed to forecast the yield of coffee in Wayanad district in Kerala. The model was validated for years 2010, 2011, and 2012 with the coffee yield data obtained during the years and the prediction was found to be good.

Analysis of multiple crystal forms of Bacillus subtilis BacB suggests a role for a metal ion as a nucleant for crystallization.

Bacillus subtilis BacB is an oxidase that is involved in the production of the antibiotic bacilysin. This protein contains two double-stranded beta-helix (cupin) domains fused in a compact arrangement. BacB crystallizes in three crystal forms under similar crystallization conditions. An interesting observation was that a slight perturbation of the crystallization droplet resulted in the nucleation of a different crystal form. An X-ray absorption scan of BacB suggested the presence of cobalt and iron in the crystal. Here, a comparative analysis of the different crystal forms of BacB is presented in an effort to identify the basis for the different lattices. It is noted that metal ions mediating interactions across the asymmetric unit dominate the different packing arrangements. Furthermore, a normalized B-factor analysis of all the crystal structures suggests that the solvent-exposed metal ions decrease the flexibility of a loop segment, perhaps influencing the choice of crystal form. The residues coordinating the surface metal ion are similar in the triclinic and monoclinic crystal forms. The coordinating ligands for the corresponding metal ion in the tetragonal crystal form are different, leading to a tighter packing arrangement. Although BacB is a monomer in solution, a dimer of BacB serves as a template on which higher order symmetrical arrangements are formed. The different crystal forms of BacB thus provide experimental evidence for metal-ion-mediated lattice formation and crystal packing.

Conformational studies suggest that the double stranded beta helix scaffold provides an optimal balance between protein stability and function.

Proteins with the double stranded beta-helix (DSBH, also known as cupin) fold perform a diverse range of functions. In this study, Bacillus subtilis quercetinase was used as a model system to understand the conformational determinants of functional diversity within the cupin fold. Controlled proteolysis experiments revealed that this enzyme is active, thermo-stable and maintains its quaternary arrangement even after substantial (ca 33 %) cleavage of the protein. The results presented in this manuscript thus show that the DSBH scaffold offers a novel balance between protein stability and function by locating the active site and substrate recognition features in the most stable region of the protein.

Crystallization and preliminary X-ray diffraction studies on the bicupin YwfC from Bacillus subtilis.

A central tenet of evolutionary biology is that proteins with diverse biochemical functions evolved from a single ancestral protein. A variation on this theme is that the functional repertoire of proteins in a living organism is enhanced by the evolution of single-chain multidomain polypeptides by gene-fusion or gene-duplication events. Proteins with a double-stranded beta-helix (cupin) scaffold perform a diverse range of functions. Bicupins are proteins with two cupin domains. There are four bicupins in Bacillus subtilis, encoded by the genes yvrK, yoaN, yxaG and ywfC. The extensive phylogenetic information on these four proteins makes them a good model system to study the evolution of function. The proteins YvrK and YoaN are oxalate decarboxylases, whereas YxaG is a quercetin dioxygenase. In an effort to aid the functional annotation of YwfC as well as to obtain a complete structure-function data set of bicupins, it was proposed to determine the crystal structure of YwfC. The bicupin YwfC was crystallized in two crystal forms. Preliminary crystallographic studies were performed on the diamond-shaped crystals, which belonged to the tetragonal space group P422. These crystals were grown using the microbatch method at 298 K. Native X-ray diffraction data from these crystals were collected to 2.2 A resolution on a home source. These crystals have unit-cell parameters a = b = 68.7, c = 211.5 A. Assuming the presence of two molecules per asymmetric unit, the V(M) value was 2.3 A3 Da(-1) and the solvent content was approximately 45%. Although the crystals appeared less frequently than the tetragonal form, YwfC also crystallizes in the monoclinic space group P2(1), with unit-cell parameters a = 46.7, b = 106.3, c = 48.7 A, beta = 92.7 degrees.

Old fold in a new X-ray diffraction dataset? Low-resolution molecular replacement using representative structural templates can provide phase information.

The advent of structural genomics has led to a dramatic increase in the number of structures deposited in the Protein Data Bank. The number of new folds, however, still remains a very small fraction of the total number of deposited structures. Recent data on the progress of the structural genomics initiative reveals that more than 85% of target proteins that progress to the stage of data collection and structure determination have a known fold. Enzymes, which tend to exploit reaction space while adopting a common stable scaffold, contribute significantly to this observation. Herein, we evaluate a method to examine the "old fold in a new dataset" scenario likely to be encountered in the structural genomics pipeline. We demonstrate that a fold detection strategy based on secondary structure signatures followed by molecular replacement using a minimalist model can be effectively used to solve the phase problem in X-ray crystallography without further recourse to heavy atom derivatives or multiple anomalous dispersion techniques. Three common folds-the triosephosphate isomerase (TIM), adenine nucleotide alpha hydrolase-like (HUP), and RNA recognition motif (RRM)-were examined using this approach. The results presented herein also provide an estimate of the extent of phase information that can be derived from a single domain in a large multidomain structure.

Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae.

Hcs77 is a putative cell surface sensor for cell integrity signaling in Saccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39 degrees C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated the MID2 gene as a dosage suppressor of the cell lysis defect of an hcs77 null mutant. MID2 encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog of MID2, designated MTL1, which provides a partially redundant function with MID2 for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.

Kinetic mechanism of nicotinic acid phosphoribosyltransferase: implications for energy coupling.

Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) is a facultative ATPase that uses the energy of ATP hydrolysis to drive the synthesis of nicotinate mononucleotide and pyrophosphate from nicotinic acid (NA) and phosphoribosyl pyrophosphate (PRPP). To learn how NAPRTase uses this hydrolytic energy, we have further delineated the kinetic mechanism using steady-state and pre-steady-state kinetics, equilibrium binding, and isotope trapping. NAPRTase undergoes covalent phosphorylation by bound ATP at a rate of 30 s-1. The phosphoenzyme (E-P) binds PRPP with a KD of 0.6 microM, a value 2000-fold lower than that measured for the nonphosphorylated enzyme. The minimal rate constant for PRPP binding to E-P is 0.72 x 10(5) M-1 s-1. Isotope trapping shows that greater than 90% of bound PRPP partitions toward product upon addition of NA. Binding of NA to E-P.PRPP is rapid, kon >/= 7.0 x 10(6) M-1 s-1, and is followed by rapid formation of NAMN and PPi, k >/= 500 s-1. After product formation, E-P undergoes hydrolytic cleavage, k = 6.3 s-1, and products NAMN, PPi, and Pi are released. Quenching from the steady state under Vmax conditions indicates that slightly less than half the enzyme is in phosphorylated forms. To account for this finding, we propose that one step in the release of products is as slow as 5.2 s-1 and, together with the E-P cleavage step, codetermines the overall kcat of 2.3 s-1 at 22 degrees C. Energy coupling by NAPRTase involves two strategies frequently proposed for ATPases of macromolecular recognition and processing. First, E-P has a 10(3)-fold higher affinity for substrates than does nonphosphorylated enzyme, allowing the E-P to bind substrate from low concentration and nonphosphorylated enzyme to expel products against a high concentration. Second, the kinetic pathway follows "rules" [Jencks, W. P. (1989) J. Biol. Chem. 264, 18855-18858] that minimize unproductive alternative reaction pathways. However, an analysis of reaction schemes based on these strategies suggests that such nonvectorial reactions are intrinsically inefficient in ATP use.

Conversion of a cosubstrate to an inhibitor: phosphorylation mutants of nicotinic acid phosphoribosyltransferase.

Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) forms nicotinic acid mononucleotide (NAMN) and PPi from 5-phosphoribosyl 1-pyrophosphate (PRPP) and nicotinic acid (NA). The Vmax NAMN synthesis activity of the Salmonella typhimurium enzyme is stimulated about 10-fold by ATP, which, when present, is hydrolyzed to ADP and Pi in 1:1 stoichiometry with NAMN formed. The overall NAPRTase reaction involves phosphorylation of a low-affinity form of the enzyme by ATP, followed by generation of a high-affinity form of the enzyme, which then binds substrates and produces NAMN. Hydrolysis of E-P then regenerates the low-affinity form of the enzyme with subsequent release of products. Our earlier studies [Gross, J., Rajavel, M., Segura, E., and Grubmeyer, C. (1996) Biochemistry 35, 3917-3924] have shown that His-219 becomes phosphorylated in the N1 (pi) position by ATP. Here, we have mutated His-219 to glutamate and asparagine and determined the properties of the purified mutant enzymes. The mutant NAPRTases fail to carry out ATPase, autophosphorylation, or ADP/ATP exchanges seen with wild-type (WT) enzyme. The mutants do catalyze the slow formation of NAMN in the absence of ATP with rates and KM values similar to those of WT. In striking contrast to WT, NAMN formation by the mutant enzymes is competitively inhibited by ATP. Thus, the NAMN synthesis reaction may occur at a site overlapping that for ATP. Previous studies suggest that the yeast NAPRTase does not catalyze NAMN synthesis in the absence of ATP. We have cloned, overexpressed, and purified the yeast enzyme and report its kinetic properties, which are similar to those of the bacterial enzyme.

Limited proteolysis of Salmonella typhimurium nicotinic acid phosphoribosyltransferase reveals ATP-linked conformational change.

Nicotinic acid phosphoribosyltransferase (NAPRTase;EC 2.4.2.11) couples stoichiometric ATP hydrolysis with formation of nicotinate mononucleotide (NAMN) from nicotinic acid and alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP). Trypsin rapidly inactivated the ATPase and NAMN synthesis activities of NAPRTase in parallel, with cleavages at Arg-384 and Lys-374 of the 399-residue protein. ATP and PRPP each provided protection against tryptic cleavage. Limited chymotryptic proteolysis of NAPRTase exhibited very similar behavior, with specific cleavage at Phe-382 and protection by substrates. Results suggest that a solvent-exposed loop encompassing Lys-374, Phe-382, and Arg-384 is protected by ATP- or PRPP-induced conformational changes. The ability of ATP to protect even under conditions in which enzyme phosphorylation was prevented by EDTA provides evidence for a distinct ATP-induced protein conformation that acts as an intermediate in energy coupling.

Energy coupling in Salmonella typhimurium nicotinic acid phosphoribosyltransferase: identification of His-219 as site of phosphorylation.

Energy coupling between ATP hydrolysis and other enzyme reactions requires the phosphorylation of substrate-derived intermediates, or the existence of enzyme-derived intermediates capable of storage and transfer of energy. Salmonella typhimurium nicotinic acid phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) couples net ATP hydrolysis to formation of NAMN and PPi from alpha-PRPP and nicotinic acid [Vinitsky, A., & Grubmeyer, C (1993) J. Biol. Chem. 268, 26004-26010]. In the current work, we have determined that the enzyme reacts with ATP to produce a covalently phosphorylated form of the enzyme (E-P), which is common to both the ATPase and NAMN synthesis functions of NAPRTase. We have isolated E-P and verified its catalytic competence. E-P showed acid lability and base stability, diagnostic of a phosphoramidate linkage. Pyridine and hydroxylamine-catalyzed hydrolysis of E-P gave second-order rate constants consistent with published values for phosphohistidine. Two-dimensional thin-layer chromatography of alkaline-hydrolyzed E-32P showed that the phosphorylated residue co-migrated with authentic 1-phosphohistidine. Chymotrypsin and trypsin proteolysis followed by HPLC and peptide sequencing localized the phosphopeptide to Ala-210 to Phe-222 of the 399-residue protein. This peptide contains a single histidine residue, His-219. NAPRTase phosphorylated at His-219 is an intermediate in the energy transduction mechanism of NAPRTase.