Gene expression profile for<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> in the early phase of flooding stress

Although flooding can often severely damage crop yields, few studies of this stress have been made at the genetic level. To identify the genes that probably function in plants at the onset of flooding stress, we constructed a cDNA library representing tobacco plants that experienced short-term stress, i.e., 2 to 4 h of submergence while under illumination. Differential screening of that library produced 73 cDNA clones that showed preferential hybridization with the probe prepared from these stressed plants. The cDNA inserts were isolated from the vector by restriction digest and subjected to reverse northern analysis, which confirmed preferential expression of 41 genes. The remainder either had no significant increase in expression under flooding stress or exhibited no identifiable signal. We then performed northern blot analyses for some selected genes to provide supporting evidence that strongly paralleled our results from the reverse-northern evaluation. Photosynthesis-related genes were the major group, followed by those for well-known glycolysis enzymes and fermentation enzymes. Other genes include those for hydrolytic enzymes and components of the ethylene synthesis pathway. Although many others also were induced, their functions could not be characterized here.     

Identification of Down-regulated Genes Modulated by an Alternative Oxidase Pathway Under Cold Stress Conditions in Watermelon Plants

This study investigated the cold responses of watermelon plants modulated by the alternative oxidase (AOX) pathway. Salicylhydroxamic acid was used in watermelon (Citrullus lanatus) plants in order to chemically inhibit the AOX pathway. After exposure to cold temperature (10°C) for 24 h, the SHAM-treated plants showed more serious cold-induced damage compared to non-treated plants. To investigate how the AOX pathway modulated the watermelon plants’ responses to cold, a polymerase chain reaction (PCR)-based suppression subtractive hybridization technique was used to isolate genes differently expressed between cold-stressed leaves of SHAM-treated and non-treated watermelon germplasm “IVSM No. 9”. After sequencing 400 randomly chosen cDNA clones, 314 uniquely expressed sequence tags (ESTs) were obtained. A total of 114 ESTs encoded unknown proteins or had no similarity in the database. Identified genes with significant protein homology mainly participated in stress and defense responses, metabolism and energetic processes, regulation of gene expression, signal transduction, protein fate and synthesis, cellular transport and others. These results indicated that watermelon plants could not respond appropriately to cold stress and suffer greater damage with an inhibited AOX pathway, thus, the AOX pathway had an important role in defining the plants cold responses.     

Transcriptional profiles of primary metabolism and signal transduction-related genes in response to water stress in field-grown sunflower genotypes using a thematic cDNA microarray

A sunflower cDNA microarray containing about 800 clones covering major metabolic and signal transduction pathways was used to study gene expression profiles in leaves and embryos of drought-tolerant and -sensitive genotypes subjected to water-deficit stress under field conditions. Using two-step ANOVA normalization and analysis models, we identified 409 differentially expressed genes among genotypes, water treatment and organs. The majority of the cDNA clones differentially expressed under water stress was found to display opposite gene expression profiles in drought-tolerant genotype compared to drought-sensitive genotype. These dissimilarities suggest that the difference between tolerant and non-tolerant plants seems to be associated with changes in qualitative but not quantitative mRNA expression. Comparing leaves and embryos, 82 cDNA clones showing organ-specific variation in gene expression levels were identified in response to water stress across genotypes. Genes related to amino acids and carbohydrates metabolisms, and signal transduction were induced in embryos and repressed in leaves; suggesting that vegetative and reproductive organs respond differentially to water stress. Adaptive mechanisms controlling water deficit tolerance are proposed and discussed.     

Cloning of cDNAs for genes that are early-responsive to dehydration stress (ERDs) inArabidopsis thaliana L.: identification of three ERDs as HSP cognate genes

InArabidopsis thaliana L., accumulation of abscisic acid (ABA) began to increase 2 h after plants had been subjected to dehydration stress and reached maximum levels after 10h. Differential hybridization was used to isolate 26Arabidopsis cDNAs with gene expression induced by a 1 h dehydration treatment. The cDNA clones were classified into 16 groups based on Southern blot hybridization, and named ERD (early-responsive todehydration) clones. Partial sequencing of the cDNA clones revealed that three ERDs were identical to those of HSP cognates (Athsp70-1, Athsp81-2, and ubiquitin extension protein). Dehydration stress strongly induced the expression of genes for the three ERDs, while application of ABA, which is known to act as a signal transmitter in dehydration-stressed plants, did not significantly affect the ERD gene expression. This result suggests that these HSP cognates are preferentially responsive to dehydration stress inA. thaliana, and that signaling pathways for the expression of these genes under conditions of dehydration stress are not mainly mediated by ABA. We also discuss the possible functions of these three ERD gene products against dehydration stress.     

EFFECTS OF FLOODING AND NUTRIENT ENRICHMENT ON BIOMASS ALLOCATION IN ACER RUBRUM SEEDLINGS

A greenhouse experiment was conducted on Acer rubrum seedlings to evaluate the effects of flood frequency on production and allocation of biomass and to test the effects of N and P fertilization on production and allocation. Seedlings from the Dismal Swamp were subjected to three flood treatments (no flooding, intermittent flooding, and continuous flooding) and four enrichment treatments (no enrichment, N additions, P additions, and N + P additions). More continuous flooding resulted in less biomass production. Biomass increased during the study in all treatments except for root mass in the continuously flooded treatment. However, production of abundant adventitious roots compensated for the lack of normal root growth. Root/shoot ratios exhibited the greatest decreases in the continuously flooded plants. Plants with N + P added had significantly more leaf, stem, and total mass than the nonenriched plants four months into the study. The N + P additions had apparently compensated for the effects of flood stress in the continuously flooded plants by the end of the study. The fertilized seedlings accumulated higher concentrations of N and P, but their nutrient use efficiency (biomass production per unit nutrient absorbed) was lower than in the nonenriched plants. Acer rubrum seedlings survive flooded conditions through several adaptations; however, theirgrowth is slowed by continuous flooding.     

A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.     

Expressed sequence tag analysis of Antarctic hairgrass Deschampsia antarctica from King George Island, Antarctica

Deschampsia antarctica is the only monocot that thrives in the tough conditions of the Antarctic region. It is an invaluable resource for the identification of genes associated with tolerance to various environmental pressures. In order to identify genes that are differentially regulated between greenhouse-grown and Antarctic field-grown plants, we initiated a detailed gene expression analysis. Antarctic plants were collected and greenhouse plants served as controls. Two different cDNA libraries were constructed with these plants. A total of 2,112 cDNA clones was sequenced and grouped into 1,199 unigene clusters consisting of 243 consensus and 956 singleton sequences. Using similarity searches against several public databases, we constructed a functional classification of the ESTs into categories such as genes related to responses to stimuli, as well as photosynthesis and metabolism. Real-time PCR analysis of various stress responsive genes revealed different patterns of regulation in the different environments, suggesting that these genes are involved in responses to specific environmental factors.     

Differential expression of ferritin genes in response to abiotic stresses and hormones in pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>)

In this study, the expression patterns of four ferritin genes (PpFer1, PpFer2, PpFer3, and PpFer4) in pear were investigated using quantitative real-time PCR. Analysis of tissue-specific expression revealed higher expression level of these genes in leaves than in other tested tissues. These ferritin genes were differentially expressed in response to various abiotic stresses and hormones treatments. The expression of ferritin wasn’t affected by Fe(III)-citrate treatment. Abscisic acid significantly enhanced the expression of all four ferritin genes, especially PpFer2, followed by N-benzylyminopurine, gibberellic acid, and indole-3-acetic acid. The expression peaks of PpFer1 and PpFer3 in leaves appeared at 6, 6, and 12 h, respectively, after pear plant was exposed to oxidative stress (5 mM H₂O₂), salt stress (200 mM NaCl), and heat stress (40°C). A significant increase in PpFer4 expression was detected at 6 h after salt stress or heat stress. The expression of ferritin genes was not altered by cold stress. These results suggested that ferritin genes might be functionally important in acclimation of pear to salt and oxidative stresses. Hormone treatments had no significant effect on expression of ferritin genes compared to abiotic stresses. This showed accumulation of ferritin genes could be operated by different transduction pathways under abiotic stresses and hormones treatments.     

Genes induced by WDS are differentially expressed in two populations of aleppo pine (<Emphasis Type="Italic">Pinus halepensis</Emphasis>)

Gene expression in response to water-deficit stress (WDS) is a critical factor determining the survival and growth of pine seedlings. To understand how gene expression varies with different water stress levels, we differentially screened a cDNA library from roots of aleppo pine (Pinus halepensis) seedlings subjected to WDS. We found 156 clones of genes up-regulated and 56 down-regulated in response to WDS. Of the 14 clones selected for further characterization, 8 resemble WDS-responsive genes previously identified in angiosperms. The genes showing similarities to known proteins included an aldehyde dehydrogenase, a late embryogenesis abundant (LEA) protein, a chitinase, a cyclophilin, an MYB factor, an inorganic pyrophosphatase, a sucrose synthase, and a 4-coumarate ligase. Six of the clones did not have any similarity to previously identified proteins of known function. Quantitative polymerase chain reaction (qPCR) was used to compare the differential expression of these genes during control (no stress), moderate, and severe WDS treatments of seedling progeny from two different geographic origins within Israel, Yirka, and Beit Jann. The difference in expression between the treatments for various genes ranged from 1.9 to 8.0 cycle threshold. Most of the genes were expressed at similar levels in trees from the two populations or at higher levels in trees from Beit Jann, Israel. Northern blot analyses done for four highly expressed genes verify the results of the qPCR.     

Expression patterns in soybean resistant to Phakopsora pachyrhizi reveal the importance of peroxidases and lipoxygenases

Soybean rust caused by Phakopsora pachyrhizi Sydow is a devastating foliar disease that has spread to most soybean growing regions throughout the world, including the USA. Four independent rust resistance genes, Rpp1–Rpp4, have been identified in soybean that recognize specific isolates of P. pachyrhizi. A suppressive subtraction hybridization (SSH) complementary DNA (cDNA) library was constructed from the soybean accession PI200492, which contains Rpp1, after inoculation with two different isolates of P. pachyrhizi that result in susceptible or immune reactions. Both forward and reverse SSH were performed using cDNA from messenger RNA pooled from 1, 6, 12, 24, and 48 h post-inoculation. A total of 1,728 SSH clones were sequenced and compared to sequences in GenBank for similarity. Microarray analyses were conducted on a custom 7883 soybean-cDNA clone array encompassing all of the soybean-rust SSH clones and expressed sequence tags from four other soybean cDNA libraries. Results of the microarray revealed 558 cDNA clones differentially expressed in the immune reaction. The majority of the upregulated cDNA clones fell into the functional category of defense. In particular, cDNA clones with similarity to peroxidases and lipoxygenases were prevalent. Downregulated cDNA clones included those with similarity to cell-wall-associated protein, such as extensins, proline-rich proteins, and xyloglucan endotransglycosylases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.