Behzad Ghareyazie

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.

Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.)

BackgroundSomatic embryogenesis (SE) is a complex biological process that occurs under inductive conditions and causes fully differentiated cells to be reprogrammed to an embryo like state. In order to get a better insight about molecular basis of the SE in Crocus sativus L. and to characterize differentially accumulated proteins during the process, a proteomic study based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry has been carried out.ResultsWe have compared proteome profiles of non-embryogenic and embryogenic calli with native corm explants. Total soluble proteins were phenol-extracted and loaded on 18 cm IPG strips for the first dimension and 11.5% sodium dodecyl sulfate-polyacrylamide gels for the second dimension. Fifty spots with more than 1.5-fold change in abundance were subjected to mass spectrometry analysis for further characterization. Among them 36 proteins could be identified, which are classified into defense and stress response, protein synthesis and processing, carbohydrate and energy metabolism, secondary metabolism, and nitrogen metabolism.ConclusionOur results showed that diverse cellular and molecular processes were affected during somatic to embryogenic transition. Differential proteomic analysis suggests a key role for ascorbate metabolism during early stage of SE, and points to the possible role of ascorbate-glutathione cycle in establishing somatic embryos.

Identification of QTLs for rice grain size and shape of Iranian cultivars using SSR markers

Grain size is a main component of rice appearance quality. In this study, we performed the SSR mapping of quantitative trait loci (QTLs) controlling grain size (grain length and breadth) and shape (length/breadth ratio) using an F2 population of a cross between two Iranian cultivars, Domsephid and Gerdeh, comprising of 192 individuals. A linkage map with 88 markers was constructed, which covered 1367.9 cM of the rice genome with an average distance of 18 cM between markers. Interval mapping procedure was used to identify the QTLs controlling three grain traits, and QTLs detected were further confirmed using composite interval mapping. A total of 11 intervals carrying 18 QTLs for three traits were identifed, that included five QTLs for grain length, seven QTLs for grain breadth, and six QTLs for grain shape. A major QTL for grain length was detected on chromosome 3, that explained 19.3% of the phenotypic variation. Two major QTLs for grain breadth were mapped on chromosomes 3 and 8, which explained 34.1% and 20% of the phenotypic variation, respectively. Another two major QTLs were identified for grain shape on chromosomes 3 and 8, which accounted for 27.1% and 20.5% of the phenotypic variance, respectively. The two QTLs that were mapped for grain shape coincided with the major QTLs detected for grain length and grain breadth. Intrestingly, gs2 QTL specific to grain shape was detected on chromosome 2 that explained 15% of the phenotypic variation.

A rapid and efficient method for regeneration of plantlets from embryo explants of cumin (Cuminum cyminum)

A new, simple and efficient method was developed for multiple shoot regeneration of cumin from imbibed embryo cultures. This method yielded a large number of shoots within short period of time (30–50 days) without any subculturing. The effects of different media, different embryo explants and various combinations of plant growth regulators (PGRs) on callus formation and shoot regeneration in cumin were investigated. Simultaneous callus formation and shoot regeneration was obtained. The best response for multiple shoot regeneration was observed on B5 medium containing 1.0 mg l−1 BAP, 0.2 mg l−1 NAA and 0.4 mg l−1 IAA, with an average of 140 shoots per explant.

Classification of rice germplasm. I. Analysis using ALP and PCR-based RFLP

The potential of using a PCR-based approach to detect DNA polymorphism for rice germplasm classification was compared with that of Southern-based RFLP analysis. Thirty-five Iranian rice varieties were studied along with 2 typical Indica and 3 typical Japonica varieties. Thirteen mapped RFLP markers were used as hybridization probes against Southern blots containing digests of one restriction endonuclease; 12 of the 13 probes detected polymorphism in the varieties. Fifteen sets of oligonucleotides derived from sequences near the ends of the same probes and of two other mapped probes were used as primers for PCR amplification of total genomic DNA of the varieties. Amplicon length polymorphisms (ALPs) were detected with 6 of the 15 sets of primers. To identify additional polymorphism, the PCR products were digested with nine different restriction endonucleases recognizing 4- or 5-bp DNA sequences and analyzed by gel electrophoresis in agarose and polyacrylamide. RFLPs were detected for 11 sets of primers, due to point mutations and to addition/deletion events that were too small to be detected as ALPs. Because PCR products are easily generated and may be analyzed in detail through the use of restriction endonucleases that cut rice DNA frequently, PCR-based RFLP analysis is a useful tool for the classification of rice germplasm.

Evidence for regional diversity and host adaptation in Iranian populations of the Russian wheat aphid

Effective pest management is greatly facilitated by knowledge of the genetic structure and host adaptation of the pest species in question. The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko) (Homoptera: Aphididae: Macrosiphini), is an important economic pest in many cereal‐growing areas of the world, and in this study we investigated these aspects of its populations, using microsatellite markers and host plant response assays. Diuraphis noxia was sampled from 38 locations in Iran and genotyped at four polymorphic microsatellite loci that had been isolated from various Sitobion species. We identified 50 multilocus genotypes in 376 individuals. The overall observed heterozygosity was 0.134. F‐statistics showed a regional partitioning in D. noxia populations with overall FST = 0.231. In addition, there was a significant correlation between genetic and geographic distances. In order to test for the ecological consequences of genetic variability in D. noxia, biotypic variation amongst the isolates collected from wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) was evaluated on a number of resistant and susceptible wheat varieties. The plant variables we measured were damage rating (based on leaf chlorosis, leaf rolling, wilting, and death of the host plant), host plant dry weight, and root length. Damage rating was the best criterion for detecting biotypic variation in D. noxia. Discriminant analysis correctly classified the isolates in respective groups in 80–91.8% of the cases. The barley isolate showed no differences in performance on resistant and susceptible wheat, indicating a lack of gene‐by‐gene relationship with wheat plants. In contrast, wheat isolates differentially damaged the resistant and susceptible plants and showed moderate to severe virulence.

DIRECT SHOOT REGENERATION FROM MATURE EMBRYO AS A RAPID AND GENOTYPE-INDEPENDENT PATHWAY IN TISSUE CULTURE OF HETEROGENEOUS DIVERSE SETS OF CUMIN (CUMINUM CYMINUM L.) GENOTYPES

SummaryA rapid and one-step protocol for direct regeneration of shoots from cumin embryo explants has been developed. Embryo explants with shoot meristems were cultured on shoot regeneration medium for 15–22 d. After embryo culture, shoots were regenerated from the area adjacent to the region between the cotyledons and embryo axis within 2 wk, without any intermediate callus phase. Shoot proliferation and elongation were achieved on shoot regeneration medium without subculture. Among the different combinations of 6-benzylaminopurine, α-naphthaleneacetic acid (NAA), and indole-3-acetic acid (IAA) tested, 0.8 mgl−1 (4.3 μM) NAA in combination with 0.3 mgl−1 (1.71 μM) IAA in the B5 medium resulted in the most efficient direct shoot regeneration. No significant difference was detected for the number of regenerated explants when different heterogeneous endemic varieties were compared. This plant regeneration procedure was applicable to different cumin genotypes and regenerated plants were phenotypically normal.

Population genetic structure based on SSR markers in alfalfa (Medicago sativa L.) from various regions contiguous to the centres of origin of the species

differentiated using mosphological descriptors, and although such descriptions are indeed useful from a breeding perspective, they are inadequate for analysis of population genetic structure. Because alfalfa is native to northwestern Iran, a high level of genetic diversity may be expected among individual plants within and between populations. Thus, the objectives of the present study were to (a) identify polymorphic SSR markers with high informative value for the Iranian alfalfa in terms of population differentiation and identification, (b) estimate the genetic population structure, and (c) compare the genetic diversity between different geographical regions, especially across different latitudes. The average within-population genetic diversity evaluated using eight simple sequence repeat (SSR) markers ranged from 0.82 in the Kerman population (low latitudes) to 0.93 in the Ghareyonjeh population (higher latitudes). The genetic relationships among populations were analysed using cluster analysis by UPGMA algorithm based on the coancestry coefficient matrix. The higher distance between Ghareyonjeh population and others was due to the presence of a specific allele in all plants within this population. The trend of changes in genetic diversity across populations was concordant with latitude changes. The obtained results suggest that SSR markers are useful for identification of alfalfa populations, for assessing interpopulation and intrapopulation genetic diversity, and for estimation of genetic differentiation.

Genetic diversity and relationships among Pistacia species and cultivars

Iran is one of the two major centres of Pistacia diversity and the main producer of pistachios in the world. About 282 Iranian pistachio genotypes (Pistacia spp.), together with 22 foreign cultivars (P. vera), were genotyped using 10 simple sequence repeat (SSR) markers to analyse the genetic diversity and relationships among Pistacia species and cultivars. The results revealed that the genetic diversity within P. atlantica subsp. kurdica was considerably lower than in P. vera or P. khinjuk. Principal coordinate analysis revealed a clear separation between the different Pistacia spices, as well as between the Iranian and foreign cultivars. AMOVA analysis showed that the variation between the species, between different populations, and within populations accounted for 41, 9, and 50% of the total variation, respectively. The results demonstrated that the study of genetic diversity and relationships among Pistacia species and cultivars using SSR markers provides important information for the collection and conservation of pistachio germplasm. In addition, the Iranian cultivars had a broader genetic background than that of the foreign cultivars. Thus, they are very important for genetic conservation and the planning of future breeding programmes. We also determined the different levels of genetic diversity that exist between and within the species and populations and showed that gene flow occurs between the Iranian cultivars and wild-type P. vera populations. The study provides practical information that policy-makers and scientists can apply to the conservation and sustainable use of all the species studied.