Joel D. L. C. Siopongco

Proteomic analysis of rice leaves during drought stress and recovery

Three‐week old plants of rice (Oryza sativa L. cv CT9993 and cv IR62266) developed gradual water stress over 23 days of transpiration without watering, during which period the mid‐day leaf water potential declined to ∼–2.4 MPa, compared with ∼–1.0 MPa in well‐watered controls. More than 1000 protein spots that were detected in leaf extracts by proteomic analysis showed reproducible abundance within replications. Of these proteins, 42 spots showed a significant change in abundance under stress, with 27 of them exhibiting a different response pattern in the two cultivars. However, only one protein (chloroplast Cu‐Zn superoxide dismutase) changed significantly in opposite directions in the two cultivars in response to drought. The most common difference was for proteins to be up‐regulated by drought in CT9993 and unaffected in IR62266; or down‐regulated by drought in IR62266 and unaffected in CT9993. By 10 days after rewatering, all proteins had returned completely or largely to the abundance of the well‐watered control. Mass spectrometry helped to identify 16 of the drought‐responsive proteins, including an actin depolymerizing factor, which was one of three proteins detectable under stress in both cultivars but undetectable in well‐watered plants or in plants 10 days after rewatering. The most abundant protein up‐regulated by drought in CT9993 and IR62266 was identified only after cloning of the corresponding cDNA. It was found to be an S‐like RNase homologue but it lacked the two active site histidines required for RNase activity. Four novel drought‐responsive mechanisms were revealed by this work: up‐regulation of S‐like RNase homologue, actin depolymerizing factor and rubisco activase, and down‐regulation of isoflavone reductase‐like protein.

A proteomic approach to analyzing drought- and salt-responsiveness in rice

The analysis of stress-responsiveness in plants is an important route to the discovery of genes conferring stress tolerance and their use in breeding programs. Proteomic analysis provides a broad view of plant responses to stress at the level of proteins. In recent years this approach has increased in sensitivity and power as a result of improvements in two-dimensional polyacrylamide gel electrophoresis (2DE), protein detection and quantification, fingerprinting and partial sequencing of proteins by mass spectrometry (MS), bioinformatics, and methods for gene isolation. 2DE provides information on changes in abundance and electrophoretic mobility of proteins, the latter reflecting post-translational modifications such as phosphorylation and free-radical cleavage. Here we review the technical aspects of proteomics and demonstrate its use in analyzing the response of rice plants to drought and salinity. More than 2000 proteins were detected reproducibly in drought-stressed and well-watered leaves. Out of >1000 proteins that were reliably quantified, 42 proteins changed significantly in abundance and/or position. We identified several leaf proteins whose abundance increased significantly during drought and declined on re-watering. The three most marked changes were seen with actin depolymerizing factor, a homologue of the S-like ribonucleases and the chloroplastic glutathione-dependent dehydroascorbate reductase. Proteomic comparisons of salt stress-tolerant and stress-sensitive genotypes revealed numerous constitutive and stress-induced differences in root proteins. Among them was caffeoyl-CoA O-methyltransferase, an enzyme of lignin biosynthesis. The abundance of ascorbate peroxidase was much higher in salt-tolerant Pokkali than in salt-sensitive IR29 in the absence of stress.

Growth and Water Use Response of Doubled-Haploid Rice Lines to Drought and Rewatering during the Vegetative Stage

Abstract We examined the responses of doubled-haploid lines (DHLs) of rice (Oryza sativa L.) to drought and rewatering in controlled rainfed lowland conditions, to test the the hypothesis that the DHLs would permit trait comparisons with less confounding by unrelated traits than had been reported previously. IR62266 and four DHLs derived from the cross between IR62266 and CT9993 (DHL-32, 51, 54 and 79) were grown in pot experiments in the greenhouse at the IRRI, Los Baños, Philippines. Genotypic variation in leaf and tiller development, transpiration, water use efficiency, osmotic adjustment and leaf water potential was examined in relation to dry matter production. Results revealed that greater seedling vigor through continued leaf expansion in early drought was associated with greater dry matter production after rewatering. A higher water use efficiency was related to a greater increase in dry matter production during drought. Leaf water potential was correlated strongly with dry weight, not only during drought, but especially on rewatering. Therefore, we found that the ability to continue leaf expansion, higher water use efficiency, and a greater osmotic adjustment for maintenance of leaf water potential as drought progressed were desirable traits for improved performance under drought and improved ability to recover on rewatering. These relationships could be analyzed precisely using such genetically-related materials as DHLs, with less confounding effects of plant size and genetic background.

Root growth and water extraction response of doubled-haploid rice lines to drought and rewatering during the vegetative stage

Abstract Doubled haploid lines (DHLs) of rice (Oryza sativa L.) were used to examine responses to drought and rewatering in controlled rainfed lowland conditions, in order to determine whether confounding by unrelated traits would be less than has been reported previously for contrasting cultivars that differ in genetic background. IR62266 and four DHLs derived from the cross between IR62266 and CT9993 (DHL-32, -51, -54 and -79) were grown in pot experiments in the greenhouse during the 2000 dry and wet seasons at IRRI, Los Baños, Philippines. There were two water regimes (well-watered and drought). Estimated water extraction obtained by time domain reflectometry (TDR) was similar to cumulative transpiration estimated from pot weighing for each genotype. Genotypic variation was observed in root traits and water extraction, with extraction slower in DHL-32 and faster in DHL-79, especially in deeper soil layers. An upper bound relationship between water extraction from a soil layer and root length density (RLD) in that layer was readily apparent over DHLs and soil depths, suggesting a critical value of RLD for water extraction of 0.30 cm cm−3 in these conditions. Because soils in the field would not be as homogenous as the puddled soils used in these greenhouse experiments, this critical RLD for water extraction from a soil layer is a reference for ideal conditions, and requires careful validation in the field. Use of DHLs permitted comparisons with reduced confounding by genetic background, with consequent improvements in precision.

Root development, water uptake, and shoot dry matter production under water deficit conditions in two CSSLs of rice: Functional roles of root plasticity

Abstract Root traits that can contribute to drought resistance have not been clearly indentified. We examined the role of root system development in enhancing water uptake and contribution to dry matter production by using the root box-pinboard method, with which quantitative assessment of root system development and the water uptake of root are possible. Chromosome segment substitution lines CSSL45 and CSSL50, and the recurrent parent Nipponbare were grown under continuously waterlogged conditions (control), and various intensities of water deficit in root boxes. There was no significant difference among the genotypes in shoot growth and root development, while CSSL45 and CSSL50 showed greater shoot dry weight than Nipponbare under water deficit conditions. This was due to their abilities to promote root system development as compared with Nipponbare, which facilitated greater water extraction than Nipponbare, especially under the mild water deficit condition of 20–25% w/w soil moisture contents. Furthermore, the increased root length density did not exceed the estimated critical value for water uptake, which indicates that plastic root system development was functionally effective and efficient for the enhancement of water uptake under mild water deficit conditions.

Stomatal Responses in Rainfed Lowland Rice to Partial Soil Drying ; Evidence for Root Signals

Abstract The role of root signals in water deficit responses of rice (Oryza sativa L.) is important in the alternate flooding and drying conditions encountered in the rainfed lowlands, where the abundant roots in shallow soil layers may generate signals when droughted, with consequent reduction in stomatal conductance (gs) and growth, despite the likelihood of additional water in deeper soil layers. This study was conducted to confirm the presence of root signals, explore their nature and plant responses, consider the suitability of the methods, and discuss implications for adaptation under rainfed lowland drought. A split-root technique was used in greenhouse studies, whereby roots were divided into two sections: flooded and droughted. The decrease in gs and transpiration rate (Tr) due to drying of a portion of the roots, and their apparent recovery upon severing of this root portion, were consistent with the role for signals. The field study confirmed the evidence for root signals during progressive soil drying, whereby gs and Tr decreased before leaf water potential (ΨL) started to decline. The increase in leaf ABA concentration under field drought, and its strong association with soil moisture tension and gs, suggested its involvement in mediating stomatal responses during early drought in rice. The recovery in ΨL after severing of droughted roots in the greenhouse could be attributed to increased hydraulic conductance. These responses imply a role for both chemical and hydraulic signals in rice, which have important implications for adaptation and crop performance in contrasting rice ecosystems.

Constraints to High Yield of Dry-Seeded Rice in the Rainy Season of a Humid Tropic Environment

Abstract Dry seeding advances establishment and harvest of rainfed rice and may help the crop escape late-season droughts. Early establishment, however, may expose the crop to early and mid-season droughts and periods of low radiation during the grain formation phase. We conducted experiments in the 1992 and 1993 rainy seasons at Tarlac, Philippines, to investigate factors that may hinder the performance and yield of dry-seeded rice cv. IR72. The treatments included three seeding dates and three water regimes (totally rainfed, irrigated from seeding to complete emergence followed by rainfed, and fully irrigated). Drought stresses between panicle initiation and flowering, with a matric potential of -25 kPa to -60 kPa in the 0-10 cm soil layer, reduced final biomass by 20%–30%. The same stresses occurring during the vegetative stage delayed flowering 3-5 d, but did not reduce total biomass at harvest. High plant density of the dry-seeding culture (325-450 seedlings m–2) resulted in excessive vegetative growth (1600 -2200 tillers m–2 at maximum tillering stage). Inter- and intra-plant competition and low radiation (especially in typhoons) during anthesis and grain filling resulted in a high rate (40-70%) of tiller abortion, delay in flowering of later tillers, low percentage of filled spikelets (30-60%), and low yield (2.5–4.3 t ha–1), despite high biomass production (13-15 t ha–1). Selecting new varieties and devising cultural practices that ensure adequate plant population and weed competitiveness in drought years and avoid excessive vegetative growth in years with low radiation are research challenges to make full use of the potential of dry seeding to increase the productivity of rainfed lowland rice.

Stomatal responses in rainfed lowland rice to partial soil drying; comparison of two lines

Abstract Previously, we demonstrated that root tips in drying soil communicate with shoots for stomatal closure in rainfed lowland rice, despite further water being available at depth. This study examines variation between two lines in root signals.Rice lines CT9993 and IR62266 were grown in the field, and in the greenhouse with the split-root root-sever wax-layer system, to investigate their responses to mild and severe water deficit by monitoring stomatal conductance (gs), leaf water potential and leaf ABA concentration. In the greenhouse, root systems were divided, withholdingwater from one portion, and in some cases, severing the droughted portion of roots to remove the signal. Wax layers differing in strength were placed at hardpan depth. Roots of CT9993 were better able to penetrate the wax layers. IR62266 exhibited stronger responses than CT9993, with IR62266’s stomatal conductance dropping sharply under water deficit, and recovering at slower rates but less completely, when roots subjected to drying soil were severed. The greater stomatal response in IR62266 was associated with a higher leaf ABA concentration during early water deficit, which in turn was associated with its greater number of roots in drying soil. In the field, a second reduction in gs wasobserved under severe water deficit, with stronger signals in IR62266 associated with more conservative water use as soil drying intensified. To better exploit subsoil water in mild or transient water deficit, selection for reduced root signals might be warranted.

Drought-induced root plasticity of two upland NERICA varieties under conditions with contrasting soil depth characteristics

Abstract To identify differences in root plasticity patterns of two upland New Rice for Africa (NERICA) varieties, NERICA 1 and 4, in response to drought under conditions with contrasting soil profile characteristics, soil moisture gradients were imposed using a sloping bed system with depths ranging 30–65 cm and a line-source sprinkler system with a uniformly shallow soil layer of 20 cm depth. Varietal differences in shoot and root growths were identified only under moderate drought conditions, 11–18% v/v soil moisture content. Further, under moderate drought soil conditions where roots could penetrate into the deep soil layer, deep root development was greater in NERICA 4 than in NERICA 1, which contributed to maintaining dry matter production. However, under soil conditions with underground impediment to deep root development, higher shoot dry weight was noted for NERICA 1 than for NERICA 4 at 11–18% v/v soil moisture content, which was attributed to increased lateral root development in the shallow soil layer in NERICA 1. Enhanced lateral root development in the 0–20-cm soil layer was identified in NERICA 1 even under soil conditions without an impediment to deep root development; however, this did not contribute to maintaining dry matter production in upland rice. Thus, we show different root developmental traits associated with drought avoidance in the two NERICA varieties, and that desirable root traits for upland rice cultivation vary depending on the target soil environment, such as the distribution of soil moisture and root penetration resistance.

Effect of Abiotic Stresses on the Nondestructive Estimation of Rice Leaf Nitrogen Concentration

Decision support tools for non-destructive estimation of rice crop nitrogen (N) status (e.g., chlorophyll meter [SPAD] or leaf color chart [LCC]) are an established technology for improved N management in irrigated systems, but their value in rainfed environments with frequent abiotic stresses remains untested. Therefore, we studied the effect of drought, salinity, phosphorus (P) deficiency, and sulfur (S) deficiency on leaf N estimates derived from SPAD and LCC measurements in a greenhouse experiment. Linear relations between chlorophyll concentration and leaf N concentration based on dry weight ( N dw ) between SPAD values adjusted for leaf thickness and N dw and between LCC scores adjusted for leaf thickness and N dw could be confirmed for all treatments and varieties used. Leaf spectral reflectance measurements did not show a stress-dependent change in the reflectance pattern, indicating that no specific element of the photosynthetic complex was affected by the stresses and at the stress level applied. We concluded that SPAD and LCC are potentially useful tools for improved N management in moderately unfavorable rice environments. However, calibration for the most common rice varieties in the target region is recommended to increase the precision of the leaf N estimates.