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Adenosine bases of RNA can be transiently modified by the deposition of a methyl-group to form N 6 -methyladenosine (m 6 A). This adenosine-methylation is an ancient process and the enzymes involved are evolutionary highly conserved. A genetic screen designed to identify suppressors of late flowering transgenic Arabidopsis plants overexpressing the miP1a microProtein yielded a new allele of the FIONA1 (FIO1) m 6 A-methyltransferase. To characterize the early flowering phenotype of fio1 mutant plants we employed an integrative approach of mRNA-seq, Nanopore direct RNA-sequencing and meRIP-seq to identify differentially expressed transcripts as well as differentially methylated RNAs. We provide evidence that FIO1 is the elusive methyltransferase responsible for the 3’-end methylation of the FLOWERING LOCUS C ( FLC ) transcript. Furthermore, our genetic and biochemical data suggest that 3’-methylation stabilizes FLC mRNAs and non-methylated FLC is a target for rapid degradation.

The circadian clock regulates plant tissue hydraulics to synchronize water supply with environmental cycles and thereby optimize growth. A role for aquaporin water channels in these processes was suggested by circadian fluctuations in aquaporin transcript abundance. Here, we show that Arabidopsis rosette hydraulic conductivity (Kros) displays a genuine circadian rhythmicity with a peak around midday. Combined immunological and proteomic approaches revealed that phosphorylation at two C-terminal sites (Ser280, Ser283) of Plasma membrane Intrinsic Protein 2;1 (AtPIP2;1), one of major plasma membrane aquaporins in rosettes, shows circadian oscillations and is correlated with Kros. Transgenic expression of phosphodeficient and phosphomimetic forms of this aquaporin indicated AtPIP2;1 phosphorylation to be necessary but not sufficient for Kros regulation. The supporting role of 14-3-3 proteins, known to interact with and regulate phosphorylated proteins, was investigated. Individual knock-out plants for five 14-3-3 protein isoforms expressed in rosettes lacked circadian activation of Kros. Two of these (GRF4 (14-3-3Phi); GRF10 (14-3-3Epsilon)) showed direct interactions with AtPIP2;1 in the plant and upon co-expression in oocytes were able to activate AtPIP2;1, preferentially when the latter is phosphorylated at its two C-terminal sites. We propose that this regulation mechanism assists in activation of phosphorylated AtPIP2;1 during circadian regulation of Kros.

Monoterpene alcohols function in plant survival strategies, but they may cause self-toxicity to plants due to their hydrophobic and highly reactive properties. To explore the role of these compounds in plant stress responses, we assessed transgenic tobacco plants overexpressing the monoterpene alcohol geraniol synthase (GES plants). Growth, morphology and photosynthetic efficiency of GES plants were not significantly different from those of control plants (wild-type and GUS-transformed plants). While GES plants’direct defenses against herbivores or pathogens were similar to those of control plants, their indirect defense (i. e., attracting herbivore enemy Nesidiocoris tenuis) was stronger compared to that of control plants. However, GES plants were susceptible to cold stress and even more susceptible to extreme heat stress (50°C), as shown by decreased levels of sugar metabolites, invertase activity and its products (Glc and Fru), and leaf starch granules. Moreover, GES plants showed decreased transcription levels of the WRKY33 transcription factor gene and an aquaporin gene (PIP2). The results of this study show that GES plants exhibit enhanced indirect defense ability against herbivores, but conversely, GES plants exhibit hypersensitivity to heat stress due to suppressed sugar metabolism and gene regulation for thermal stress tolerance.

Cassava mosaic disease is a major constraint for cassava production in Africa, resulting in significant economic losses. We have engineered transgenic cassava with resistance to African cassava mosaic virus (ACMV), by expressing ACMV AC1-homologous hairpin double-strand RNAs. Transgenic cassava lines with high levels of AC1-homologous small RNAs have ACMV immunity with increasing viral load and different inoculation methods. We report a correlation between the expression of the AC1-homologous small RNAs and the ACMV resistance of the transgenic cassava lines. Characterization of the small RNAs revealed that only some of the hairpin-derived small RNAs fall into currently known small interfering RNA classes in plants. The method is scalable to stacking by targeting multiple virus isolates with additional hairpins.

A comprehensive mutant library is a fundamental resource for investigating gene functions, especially after the completion of genome sequencing. A new tobacco mutant population induced by ethyl methane sulfonate mutagenesis was developed for functional genomics applications. We isolated 1607 mutant lines and 8610 mutant plants with altered morphological phenotypes from 5513 independent M2 families that consisted of 69,531 M2 plants. The 2196 mutations of abnormal phenotypes in the M2 putative mutants were classified into four groups with 17 major categories and 51 subcategories. More than 60% of the abnormal phenotypes observed fell within the five major categories including plant height, leaf shape, leaf surface, leaf color, and flowering time. The 465 M2 mutants exhibited multiple phenotypes, and 1054 of the 2196 mutations were pleiotropic. Verification of the phenotypes in advanced generations indicated that 70.63% of the M3 lines, 84.87% of the M4 lines, and 95.75% of the M5 lines could transmit original mutant phenotypes of the corresponding M2, M3, and M4 mutant plants. Along with the increased generation of mutants, the ratios of lines inheriting OMPs increased and lines with emerging novel mutant phenotypes decreased. Genetic analyses of 18 stably heritable mutants showed that two mutants were double recessive, five were monogenic recessive, eight presented monogenic dominant inheritance, and three presented semi-dominant inheritance. The pleiotropy pattern, saturability evaluation, research prospects of genome, and phenome of the mutant populations were also discussed. Simultaneously, this novel mutant library provided a fundamental resource for investigating gene functions in tobacco.

Nutrition recommendations worldwide emphasize ingestion of plant-based diets rather than diets that rely primarily on animal products. However, this plant-based diet could limit the intake of essential nutrients such as calcium. Osteoporosis is one of the world's most prevalent nutritional disorders, and inadequate dietary calcium is a known contributor to the pathophysiology of this condition. Previously, we have modified carrots to express increased levels of a plant calcium transporter (sCAX1), and these plants contain [almost equal to]2-fold-higher calcium content in the edible portions of the carrots. However, it was unproven whether this change would increase the total amount of bioavailable calcium. In randomized trials, we labeled these modified carrots with isotopic calcium and fed them to mice and humans to assess calcium bioavailability. In mice feeding regimes (n = 120), we measured ⁴⁵Ca incorporation into bones and determined that mice required twice the serving size of control carrots to obtain the calcium found in sCAX1 carrots. We used a dual-stable isotope method with ⁴²Ca-labeled carrots and i.v. ⁴⁶Ca to determine the absorption of calcium from these carrots in humans. In a cross-over study of 15 male and 15 female adults, we found that when people were fed sCAX1 and control carrots, total calcium absorption per 100 g of carrots was 41% ± 2% higher in sCAX1 carrots. Both the mice and human feeding studies demonstrate increased calcium absorption from sCAX1-expressing carrots compared with controls. These results demonstrate an alternative means of fortifying vegetables with bioavailable calcium.

Abstract Genetically modified plants are one of the tactics used in integrated pest management - IPM. There is great concern about the impact of these plants on non-target organisms. On the other hand, there is little information in the literature on the effects of transgenics (Bacillus thuringiensis) Bt on populations of phytophagous mites, and the physiological responses that this attack promotes on plants. The objective of this work was to evaluate the biology of the T. ludeni mite in Bt cotton, expressing the Cry1F and Cry1Ac proteins. To evaluate the behavior of food and oviposition preference of the T. ludeni with Bt cotton and isohybrid. Verify if the physiological stress caused by T. ludeni’s attack is differentiated in Bt cotton. The mites were reared in Bt cotton and isohybrid, in a total of 40 replicates in the completely randomized design and the biological cycle was evaluated. The food preference and oviposition analysis were done with 10 replicates, with choice. The physiological stress was evaluated through chlorophyll fluorescence, under greenhouse conditions. The data of the T. ludeni biology were analyzed by Student's t-test, for food and oviposition preference the chi-square test was performed. Regression models were fitted for the fluorescence parameters. The model identity test was used to evaluate the differences between Bt and isohybrid treatments. Cry1F and Cry1Ac proteins have not affected the biology of T. ludeni. The photosynthetic parameters in Bt cotton plants were less influenced by T. ludeni infestation. Resumo O uso de plantas geneticamente modificadas é uma das táticas utilizadas no manejo integrado de pragas - MIP. Observa-se grande preocupação com o impacto dessas plantas sobre organismos não alvos. Por outro lado, existe pouca informação na literatura sobre efeitos dos transgênicos (Bacillus thuringiensis) Bt em populações de ácaros fitófagos, e as respostas fisiológicas que esse ataque promove nas plantas. Objetivou-se com esse trabalho avaliar a biologia do ácaro T. ludeni em algodoeiro Bt, expressando as proteínas Cry1F e Cry1Ac. Avaliar se há comportamento de preferência alimentar e postura de T. ludeni em relação ao algodoeiro Bt e seu iso-híbrido. E verificar se o estresse fisiológico causado pelo ataque de T. ludeni é diferenciado em algodoeiro Bt. Os ácaros foram criados em algodoeiro Bt e iso-híbrido, em um total de 40 repetições no delineamento inteiramente casualizado, onde foi avaliado o ciclo biológico. A análise de preferência alimentar e de posturas foi feita com 10 repetições, com escolha. O estresse fisiológico foi avaliando através da fluorescência da clorofila, em casa de vegetação. Os dados da biologia de T. ludeni foram analisados pelo teste t Student, para preferência alimentar e postura foi realizado o teste qui-quadrado. Para os parâmetros da fluorescência, foram ajustados modelos de regressão. Para testar as diferenças entre Bt e iso-híbrido foi utilizado o teste de identidade de modelos. As proteínas Cry1F e Cry1Ac não afetaram a biologia de T. ludeni. Os parâmetros fotossintéticos em plantas de algodoeiro Bt foram menos influenciados pela infestação de T. ludeni.

Crop leaves in full sunlight dissipate damaging excess absorbed light energy as heat. When sunlit leaves are shaded by clouds or other leaves, this protective dissipation continues for many minutes and reduces photosynthesis. Calculations have shown that this could cost field crops up to 20% of their potential yield. Here, we describe the bioengineering of an accelerated response to natural shading events in Nicotiana (tobacco), resulting in increased leaf carbon dioxide uptake and plant dry matter productivity by about 15% in fluctuating light. Because the photoprotective mechanism that has been altered is common to all flowering plants and crops, the findings provide proof of concept for a route to obtaining a sustainable increase in productivity for food crops and a much-needed yield jump.

CRISPR-Cas9 genome editing is achieved in Arabidopsis , tobacco, lettuce and rice without introducing foreign DNA into the plants. Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana , tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) family transcription factors have well-documented functions in stress responses, but their roles in brassinosteroid (BR)-regulated growth and stress responses have not been established. Here, we show that the Arabidopsis (Arabidopsis thaliana) stress-inducible AP2/ERF transcription factor TINY inhibits BR-regulated growth while promoting drought responses. TINY-overexpressing plants have stunted growth, increased sensitivity to BR biosynthesis inhibitors, and compromised BR-responsive gene expression. By contrast, tiny tiny2 tiny3 triple mutants have increased BR-regulated growth and BR-responsive gene expression. TINY positively regulates drought responses by activating drought-responsive genes and promoting abscisic acid–mediated stomatal closure. Global gene expression studies revealed that TINY and BRs have opposite effects on plant growth and stress response genes. TINY interacts with and antagonizes BRASSINOSTERIOID INSENSITIVE1-ETHYL METHANESULFONATE SUPRESSOR1 (BES1) in the regulation of these genes. Glycogen synthase kinase 3-like protein kinase BR-INSENSITIVE2 (BIN2), a negative regulator in the BR pathway, phosphorylates and stabilizes TINY, providing a mechanism for BR-mediated downregulation of TINY to prevent activation of stress responses under optimal growth conditions. Taken together, our results demonstrate that BR signaling negatively regulates TINY through BIN2 phosphorylation and TINY positively regulates drought responses, as well as inhibiting BR-mediated growth through TINY-BES1 antagonistic interactions. Our results thus provide insight into the coordination of BR-regulated growth and drought responses.