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1. chinaXiv:201605.00492 [pdf]

The tomato mutant ars1 (altered response to salt stress 1) identifies an R1‐type MYB transcription factor involved in stomatal closure under salt acclimation

Juan F. Campos; Beatriz Cara; Fernando Pérez-Martín; Benito Pineda; Isabel Egea; Francisco B. Flores; Nieves Fernandez-Garcia; Juan Capel; Vicente Moreno; Trinidad Angosto; Rafael Lozano; Maria C. Bolarin
Subjects: Biology >> Botany >> Plant physiology

A screening under salt stress conditions of a T-DNA mutant collection of tomato (Solanum lycopersicum L.) led to the identification of the altered response to salt stress 1 (ars1) mutant, which showed a salt-sensitive phenotype. Genetic analysis of the ars1 mutation revealed that a single T-DNA insertion in the ARS1 gene was responsible of the mutant phenotype. ARS1 coded for an R1-MYB type tran ARS1 did not affect this agronomic trait. The stomatal behaviour of ars1 mutant leaves induced higher Na+ accumulation via the transpiration stream, as the decreases of stomatal conductance and transpiration rate induced by salt stress were markedly lower in the mutant plants. Moreover, the mutation affected stomatal closure in a response mediated by abscisic acid (ABA). The characterization of tomato transgenic lines silencing and overexpressing ARS1 corroborates the role of the gene in regulating the water loss via transpiration under salinity. Together, our results show that ARS1 tomato gene contributes to reduce transpirational water loss under salt stress. Finally, this gene could be interesting for tomato molecular breeding, because its manipulation could lead to improved stress tolerance without yield penalty under optimal culture conditions.

submitted time 2016-05-04 Hits3073Downloads1330 Comment 0

2. chinaXiv:201605.00491 [pdf]

Enhancing blast disease resistance by overexpression of the calcium‐dependent protein kinase OsCPK4 in rice

Mireia Bundó; aría Coca
Subjects: Biology >> Botany >> Plant physiology

Rice is the most important staple food for more than half of the human population, and blast disease is the most serious disease affecting global rice production. In this work, the isoform OsCPK4 of the rice calcium-dependent protein kinase family is reported as a regulator of rice immunity to blast fungal infection. It shows that overexpression of OsCPK4 gene in rice plants enhances resistance to blast disease by preventing fungal penetration. The constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defence response, including the production of reactive oxygen species, callose deposition and defence gene expression. OsCPK4 overexpression leads also to constitutive increased content of the glycosylated salicylic acid hormone in leaves without compromising rice yield. Given that OsCPK4 overexpression was known to confer also salt and drought tolerance in rice, the results reported in this article demonstrate that OsCPK4 acts as a convergence component that positively modulates both biotic and abiotic signalling pathways. Altogether, our findings indicate that OsCPK4 is a potential molecular target to improve not only abiotic stress tolerance, but also blast disease resistance of rice crops.

submitted time 2016-05-04 Hits3047Downloads1285 Comment 0

3. chinaXiv:201605.00488 [pdf]

Stable expression of silencing‐suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Fatima Naim; Pushkar Shrestha; Surinder P. Singh; Peter M. Waterhouse; Craig C. Wood
Subjects: Biology >> Botany >> Plant physiology

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

submitted time 2016-05-04 Hits3123Downloads1273 Comment 0

4. chinaXiv:201605.00483 [pdf]

Metabolic regulation of triacylglycerol accumulation in the green algae: identification of potential targets for engineering to improve oil yield

Elton C. Goncalves; Ann C. Wilkie; Matias Kirst; Bala Rathinasabapathi
Subjects: Biology >> Botany >> Plant physiology

The great need for more sustainable alternatives to fossil fuels has increased our research interests in algal biofuels. Microalgal cells, characterized by high photosynthetic efficiency and rapid cell division, are an excellent source of neutral lipids as potential fuel stocks. Various stress factors, especially nutrient-starvation conditions, induce an increased formation of lipid bodies filled with triacylglycerol in these cells. Here we review our knowledge base on glycerolipid synthesis in the green algae with an emphasis on recent studies on carbon flux, redistribution of lipids under nutrient-limiting conditions and its regulation. We discuss the contributions and limitations of classical and novel approaches used to elucidate the algal triacylglycerol biosynthetic pathway and its regulatory network in green algae. Also discussed are gaps in knowledge and suggestions for much needed research both on the biology of triacylglycerol accumulation and possible avenues to engineer improved algal strains.

submitted time 2016-05-04 Hits3169Downloads1388 Comment 0

5. chinaXiv:201605.00478 [pdf]

Immunotherapy using algal‐produced Ara h 1 core domain suppresses peanut allergy in mice

James A. Gregory; Ariel Shepley-McTaggart; Michelle Umpierrez; Barry K. Hurlburt; Soheila J. Maleki; Hugh A. Sampson; Stephen P. Mayfield; M. Cecilia Berin
Subjects: Biology >> Botany >> Plant physiology

Peanut allergy is an IgE-mediated adverse reaction to a subset of proteins found in peanuts. Immunotherapy aims to desensitize allergic patients through repeated and escalating exposures for several months to years using extracts or flours. The complex mix of proteins and variability between preparations complicates immunotherapy studies. Moreover, peanut immunotherapy is associated with frequent negative side effects and patients are often at risk of allergic reactions once immunotherapy is discontinued. Allergen-specific approaches using recombinant proteins are an attractive alternative because they allow more precise dosing and the opportunity to engineer proteins with improved safety profiles. We tested whether Ara h 1 and Ara h 2, two major peanut allergens, could be produced using chloroplast of the unicellular eukaryotic alga, Chlamydomonas reinhardtii. C. reinhardtii is novel host for producing allergens that is genetically tractable, inexpensive and easy to grow, and is able to produce more complex proteins than bacterial hosts. Compared to the native proteins, algal-produced Ara h 1 core domain and Ara h 2 have a reduced affinity for IgE from peanut-allergic patients. We further found that immunotherapy using algal-produced Ara h 1 core domain confers protection from peanut-induced anaphylaxis in a murine model of peanut allergy.

submitted time 2016-05-04 Hits2888Downloads1220 Comment 0

6. chinaXiv:201605.00472 [pdf]

Rice endosperm is cost‐effective for the production of recombinant griffithsin with potent activity against HIV

Evangelia Vamvaka; Elsa Arcalis; Koreen Ramessar; Abbey Evans; Barry R. O'Keefe; Robin J. Shattock; Vicente Medina; Eva St?ger; Paul Christou; Teresa Capell
Subjects: Biology >> Botany >> Plant physiology

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV-endemic regions such as sub-Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of OSGRFT in the best-performing plants was 223?μg/g dry seed weight. We also established a one-step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger-scale process to facilitate inexpensive downstream processing. OSGRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole-cell assays using purified OSGRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure OSGRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom-to-operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8?kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component.

submitted time 2016-05-04 Hits3285Downloads1431 Comment 0

7. chinaXiv:201605.00464 [pdf]

The Arabidopsis transcriptional regulator DPB3‐1 enhances heat stress tolerance without growth retardation in rice

Hikaru Sato; Daisuke Todaka; Madoka Kudo; Junya Mizoi; Satoshi Kidokoro; Yu Zhao; Kazuo Shinozaki; Kazuko Yamaguchi-Shinozaki
Subjects: Biology >> Botany >> Plant physiology

The enhancement of heat stress tolerance in crops is an important challenge for food security to facilitate adaptation to global warming. In Arabidopsis thaliana, the transcriptional regulator DNA polymerase II subunit B3-1 (DPB3-1)/nuclear factor Y subunit C10 (NF-YC10) has been reported as a positive regulator of Dehydration-responsive element binding protein 2A (DREB2A), and the overexpression of DPB3-1 enhances heat stress tolerance without growth retardation. Here, we show that DPB3-1 interacts with DREB2A homologues in rice and soya bean. Transactivation analyses with Arabidopsis and rice mesophyll protoplasts indicate that DPB3-1 and its rice homologue OsDPB3-2 function as positive regulators of DREB2A homologues. Overexpression of DPB3-1 did not affect plant growth or yield in rice under nonstress conditions. Moreover, DPB3-1-overexpressing rice showed enhanced heat stress tolerance. Microarray analysis revealed that many heat stress-inducible genes were up-regulated in DPB3-1-overexpressing rice under heat stress conditions. However, the overexpression of DPB3-1 using a constitutive promoter had almost no effect on the expression of these genes under nonstress conditions. This may be because DPB3-1 is a coactivator and thus lacks inherent transcriptional activity. We conclude that DPB3-1, a coactivator that functions specifically under abiotic stress conditions, could be utilized to increase heat stress tolerance in crops without negative effects on vegetative and reproductive growth.

submitted time 2016-05-04 Hits3352Downloads1311 Comment 0

8. chinaXiv:201605.00457 [pdf]

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

Daniel Alves Ramiro; Danila Montewka Melotto-Passarin; Mariana de Almeida Barbosa; Flavio dos Santos; Sergio Gregorio Perez Gomez; Nelson Sidnei Massola Júnior; Eric Lam; Helaine Carrer
Subjects: Biology >> Botany >> Plant physiology

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor-1 from Arabidopsis thaliana (AtBI-1), can confer increased tolerance of sugarcane plants to long-term (>20?days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C4 grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long-term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.

submitted time 2016-05-04 Hits3017Downloads1307 Comment 0

9. chinaXiv:201605.00453 [pdf]

TERMINAL FLOWER1 is a breeding target for a novel everbearing trait and tailored flowering responses in cultivated strawberry (Fragaria?×?ananassa Duch.)

Elli Aurora Koskela; Anita S?nsteby; Henryk Flachowsky; Ola Mikal Heide; Magda-Viola Hanke; Paula Elomaa; Timo Hyt?nen
Subjects: Biology >> Botany >> Plant physiology

The effects of daylength and temperature on flowering of the cultivated octoploid strawberry (Fragaria?×?ananassa Duch.) have been studied extensively at the physiological level, but information on the molecular pathways controlling flowering in the species is scarce. The flowering pathway has been studied at the molecular level in the diploid short-day woodland strawberry (F.?vesca L.), in which the FLOWERING LOCUS T1 (FvFT1)–SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (FvSOC1)–TERMINAL FLOWER1 (FvTFL1) pathway is essential for the correct timing of flowering. In this work, we show by transgenic approach that the silencing of the floral repressor FaTFL1 in the octoploid short-day cultivar ‘Elsanta’ is sufficient to induce perpetual flowering under long days without direct changes in vegetative reproduction. We also demonstrate that although the genes FaFT1 and FaSOC1 show similar expression patterns in different cultivars, the regulation of FaTFL1 varies widely from cultivar to cultivar and is correlated with floral induction, indicating that the tran FaTFL1 occurs at least partially independently of the FaFT1FaSOC1 module. Our results indicate that changing the expression patterns of FaTFL1 through biotechnological or conventional breeding approaches could result in strawberries with specific flowering and runnering characteristics including new types of everbearing cultivars.

submitted time 2016-05-04 Hits2977Downloads1277 Comment 0

10. chinaXiv:201605.00451 [pdf]

Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off‐target pathways through metabolite signalling

Elise K. Pasoreck; Jin Su; Ian M. Silverman; Sager J. Gosai; Brian D. Gregory; Joshua S. Yuan; Henry Daniell
Subjects: Biology >> Botany >> Plant physiology

The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were ~4300-fold higher in C and CN lines than in N, but all accumulated ~150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65–120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. The mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering.

submitted time 2016-05-04 Hits2774Downloads1491 Comment 0

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