Molecular cloning of stress-induced genes of maize (Zea mays L.) using the pcr-select cdna subtraction technique

Environmental abiotic stresses, such as drought, high-salinity and low temperature, severely impair plant growth and development and limit crop productivity. In order to survive and adapt to these stresses, plants must induce various physiological, bichemical and molecular changes, including the adaptation of the photosynthetic apparatus, changing in the membrane lipid, the activation of calcium influxes and Ca2+-dependent protein kinase cascades, the accumulation of proline, glycine betaine, soluble sugars and increasing the levels of antioxidants. All these changes are accompanied by notable increases or decreases in the transcript level of specific genes. Hence, transcriptional control of stress-regulated genes is a crucial part of plant responses to abiotic stresses; a further characterization of such gene transcripts in plants may help us to understand the molecular basis of the plant response to abiotic stresses and to identify new targets for manipulating biochemical, physiological and developmental processes in plants. To clarify the process of the response of maize to cold stress and to discover maize genes associated with the response pathway(s), genes induced by cold treatment were isolated according to the PCR-select cDNA subtraction method. 18 cold-induced genes (ZmCOI) were detected at 6°C. They were divided into 6 groups, based on their functions. The cold induction of these genes was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. The sequences of these 18 cold-induced genes have been deposited in GenBank under accesion numbers from DQ078760 to DQ078778

Article 3: Characterization of the stress-induced gene ZmCOI6.1 in maize: Expression and promoter sequences

Using cDNA subtraction technique, 18 cold stress responsive-genes were identified, among them a novel gene, ZmCOI6.1, whose function is still unknown. Analysis of the ZmCOI6.1 promoter sequence revealed several conserved stress-responsive cis-acting elements. Further expression characterization shows that ZmCOI6.1 is induced, in addition by cold, by other  abiotic stresses such as drought and NaCl as well as by signalling molecules such as ABA and SA. The results indicate that ZmCOI6.1 is a general stress responsive gene. A possible regulation mechanism is presented where ZmCOI6.1 is alternatively spliced yielding two transcripts whose levels are changed upon different stress treatments. Furthermore the predicted ZmCOI6.1 amino acid sequence and its homologue show high similarity with proteins in rice and Arabidopsis suggesting that it belongs to a conserved protein in plant

Temperature at night affects the genetic control of acclimation to cold in maize seedlings

Although suboptimal temperatures during maize (Zea mays L) seedling growth are known to result in decreased photosynthetic efficiency due to a combination of temperature and light stress, details remain scant on the impact of low night temperatures on photosynthetic activity. To better understand the role of night temperature on the acclimation of the photosynthetic apparatus to suboptimal temperature, a QTL experiment was conducted with the IBM302 population. Seedlings were grown under optimal temperature (24/22°C, day/night) or under subopti¬mal temperatures (17°C day and 6 or 13°C night). The two parental lines, B73 and Mo17, responded somewhat differently to suboptimal temperatures, as revealed by measurements of the operating quantum efficiency of PS II (Fq’/Fm’), the maximum quantum efficiency of PS II primary photochemistry (Fv/Fm) and leaf greenness (SPAD). While Mo17 showed very little change in response to the temperature at night, B73 exhibited a lower photosyn¬thetic performance at 13°C than at 6°C at night. At 17/6°C the photosynthetic efficiency of both genotypes was similar. These observations were supported by QTL analyses. A major QTL for photosynthesis-related traits was detected on chromosome 5 with the favorable allele contributed by Mo17. This QTL showed a lower additive effect at a temperature of 6°C than at 13°C during the night and appeared to be the major factor explaining the differential response of the parental lines to changes in the temperature at night. As potential candidate genes for this locus, ivr2 (coding for an acid vacuolar invertase) and a2 (coding for an anthocyanidin synthase) were identi¬fied. QTL analyses for invertase activity and anthocyanin content revealed a QTL for invertase activity near the ivr1 gene and a QTL for anthocyanin content close to the r1 locus, both, however, were not related to the major QTL for photosynthesis-related traits. Comparative QTL analyses of photosynthetic traits of this population and other pulished studies revealed conserved QTL regions on chromosomes 6 and 8

The protein interactome of the citrus Huanglongbing pathogen Candidatus Liberibacter asiaticus

Abstract The bacterium Candidatus Liberibacter asiaticus (CLas) causes citrus Huanglongbing disease. Our understanding of the pathogenicity and biology of this microorganism remains limited because CLas has not yet been cultivated in artificial media. Its genome is relatively small and encodes approximately 1136 proteins, of which 415 have unknown functions. Here, we use a high-throughput yeast-two-hybrid (Y2H) screen to identify interactions between CLas proteins, thus providing insights into their potential functions. We identify 4245 interactions between 542 proteins, after screening 916 bait and 936 prey proteins. The false positive rate of the Y2H assay is estimated to be 2.9%. Pull-down assays for nine protein-protein interactions (PPIs) likely involved in flagellar function support the robustness of the Y2H results. The average number of PPIs per node in the CLas interactome is 15.6, which is higher than the numbers previously reported for interactomes of free-living bacteria, suggesting that CLas genome reduction has been accompanied by increased protein multi-functionality. We propose potential functions for 171 uncharacterized proteins, based on the PPI results, guilt-by-association analyses, and comparison with data from other bacterial species. We identify 40 hub-node proteins, including quinone oxidoreductase and LysR, which are known to protect other bacteria against oxidative stress and might be important for CLas survival in the phloem. We expect our PPI database to facilitate research on CLas biology and pathogenicity mechanisms

Phylogenetic and Molecular Variability Studies Reveal a New Genetic Clade of Citrus leprosis virus C

Citrus leprosis virus C (CiLV-C) causes a severe disease affecting citrus orchards in the Western hemisphere. This study reveals the molecular variability of the virus by analyzing four genomic regions (p29, p15, MP and RNA2-intergenic region) distributed over its two RNAs. Nucleotide diversity (π) values were relatively low but statistically different over the analyzed genes and subpopulations, indicating their distinct evolutionary history. Values of πp29 and πMP were higher than those of πp15 and πRNA2–IR, whereas πMP was increased due to novel discovered isolates phylogenetically clustered in a divergent clade that we called SJP. Isolate BR_SP_SJP_01 RNA1 and RNA2 sequences, clade SJP, showed an identity of 85.6% and 88.4%, respectively, with those corresponding to CiLV-C, the type member of the genus Cilevirus, and its RNA2 5′-proximal region was revealed as a minor donor in a putative inter-clade recombination event. In addition to citrus, BR_SP_SJP_01 naturally infects the weed Commelina benghalensis and is efficiently transmitted by Brevipalpus yothersi mites. Our data demonstrated that negative selection was the major force operating in the evaluated viral coding regions and defined amino acids putatively relevant for the biological function of cilevirus proteins. This work provides molecular tools and sets up a framework for further epidemiological studies