Short-day Conditions Research Articles

WRKY transcription factors WRKY12 and WRKY13 interact with SPL10 to modulate age-mediated flowering.

WRKY12 and WRKY13 are two WRKY transcription factors that play important roles in the control of flowering time under short-day (SD) conditions. The temporally regulated expression of WRKY12 and WRKY13 indicates that they may be involved in the age-mediated flowering pathway. However, their roles in this pathway are poorly understood. Here, we show that the transcription of WRKY12 and WRKY13 is directly regulated by SQUAMOSA PROMOTER BINDING-LIKE 10 (SPL10), a transcription factor downstream of the age pathway. Binding and activation analyses revealed that SPL10 functions as a positive regulator of WRKY12 and a negative regulator of WRKY13. Further mechanistic investigation revealed that WRKY12 and WRKY13 physically interact with SPL10 and that both of them bind to the promoter of miR172b. Thus, the WRKY12-SPL10 and WRKY13-SPL10 interactions facilitate and inhibit SPL10 transcriptional function, respectively, to regulate miR172b expression. Together, our results show that WRKY12 and WRKY13 participate in the control of age-mediated flowering under SD conditions though physically interacting with SPLs and co-regulating the target gene miR172b.

Cryptic Haploid Stages in the Life Cycle of Leathesia marina (Chordariaceae, Phaeophyceae) Under In Vitro Culture.

We evaluated the life cycle of Leathesia marina through molecular analyses, culture studies, morphological observations, and ploidy measurements. Macroscopic sporophytes were collected from two localities in Atlantic Patagonia and were cultured under long-day (LD) and short-day (SD) conditions. Molecular identification of the microscopic and macroscopic phases was performed through the cox3 and rbcL genes and the phylogeny was assessed on the basis of single gene and concatenated datasets. Nuclear ploidy of each phase was estimated from the DNA contents of individual nuclei through epifluorescence microscopy and flow cytometry. Molecular results confirmed the identity of the Argentinian specimens as L.marina and revealed their conspecificity with L.marina from New Zealand, Germany, and Japan. The sporophytic macrothalli (2n) released mitospores from plurilocular sporangia, which developed into globular microthalli (2n), morphologically similar to the sporophytes but not in size, constituting a generation of small diploid thalli, with a mean fluorescent nuclei cross-sectional area of 3.21±0.7μm2 . The unilocular sporangia released meiospores that developed two morphologically different types of microthalli: erect branched microthalli (n) with a nuclear area of 1.48±0.07µm2 that reproduces asexually, and prostrate branched microthalli (n) with a nuclear area of 1.24±0.10µm2 that reproduces sexually. The prostrate microthalli released gametes in LD conditions, which merged and produced macroscopic thalli with a nuclear cross-sectional area of 3.45 ±0.09µm2 . Flow cytometry confirmed that the erect and prostrate microthalli were haploid and that the globular microthalli and macrothalli were diploid.

FRS7 and FRS12 recruit NINJA to regulate expression of glucosinolate biosynthesis genes.

The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation.

MRG1/2 histone methylation readers and HD2C histone deacetylase associate in repression of the florigen gene FT to set a proper flowering time in response to day-length changes.

Day-length changes represent an important cue for modulating flowering time. In Arabidopsis, the expression of the florigen gene FLOWERING LOCUS T (FT) exhibits a 24-h circadian rhythm under long-day (LD) conditions. Here we focus on the chromatin-based mechanism regarding the control of FT expression. We conducted co-immunoprecipitation assays along with LC-MS/MS analysis and identified HD2C histone deacetylase as the binding protein of the H3K4/H3K36 methylation reader MRG2. HD2C and MRG1/2 regulate flowering time under LD conditions, but not under short-day conditions. Moreover, HD2C functions as an effective deacetylase in planta, mainly targeting H3K9ac, H3K23ac and H3K27ac. At dusk, HD2C is recruited to FT to deacetylate histones and repress transcription in an MRG1/2-dependent manner. More importantly, HD2C competes with CO for the binding of MRG2, and the accumulation of HD2C at the FT locus occurs at the end of the day. Our findings not only reveal a histone deacetylation mechanism contributing to prevent FT overexpression and precocious flowering, but also support the model in which the histone methylation readers MRG1/2 provide a platform on chromatin for connecting regulatory factors involved in activating FT expression in response to daylight and decreasing FT expression around dusk under long days.

Photoperiod-Specific Expression of Eyes Absent 3 Splice Variant in the Pars Tuberalis of the Japanese Quail.

The molecular mechanism underlying photoperiodic response in seasonal breeding animals such as the Japanese quail, red jungle fowl, sheep, mouse, and hamster involves thyroid-stimulating hormone beta subunit (TSHβ) mRNA expression in the pars tuberalis stimulated by the extension in day length. Furthermore, this mechanism is regulated by eyes absent 3 (Eya3) in mammals. Even in birds, the expression of both TSHβ and EYA3 is induced in the pars tuberalis by the extension in day length; however, the relationship between the two genes is unknown. To clarify the function of EYA3 in quail photoperiodism, in the present study, we performed mRNA structure analysis of the Japanese quail EYA3 mRNA using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. The results revealed that there are four types of splice variants within regions of exons 7, 8, and 9 of quail EYA3 mRNA. Among the four splice variants of quail EYA3, the splice variant containing exon 7 was expressed in the pars tuberalis on the first long day, when quails were transferred from the short-day condition to the long-day condition. The results indicate that EYA3 splice variant containing exon 7 is involved in the photoperiodic response of the pars tuberalis in the Japanese quail.

RFRP3 increases food intake in a sex-dependent manner in the seasonal hamster Phodopus sungorus.

In addition to its regulatory role in luteinising hormone secretion, Rfamide-related peptide 3 (RFRP3) has also been reported to modulate food intake in several mammalian species. Djungarian hamsters (Phodopus sungorus), similar to other seasonal mammals, display a remarkable inhibition of RFRP3 expression in winter short-day conditions, associated with decreased food intake and bodyweight. This species is therefore a valuable model for assessing whether RFRP3 might be involved in the seasonal control of feeding behaviour and investigating its possible brain targets. We found that, although both male and female animals exhibit the same robust reduction in Rfrp expression in short- (SD) compared to long-day (LD) conditions, acute central administration of RFRP3 displays sex-dependent effects on food intake. RFRP3 increased food intake in female hamsters in SD or in LD dioestrus, but not in LD pro-oestrus, indicating that the orexigenic effect of RFRP3 is observed in conditions of low circulating oestradiol levels. In male hamsters, food intake was not changed by acute injections of RFRP3, regardless of whether animals were in SD or LD conditions. Analysing the gene expression of various metabolic neuropeptides in the brain of RFRP3-injected Djungarian hamsters revealed that Npy expression was increased in female but not in male animals. The present study suggests that, in Djungarian hamsters, RFRP3 exhibits a sex-dependent orexigenic effect possibly by inducing increased Npy expression.

Phosphatidic acid directly binds with rice potassium channel OsAKT2 to inhibit its activity.

The plant Shaker K+ channel AtAKT2 has been identified as a weakly rectifying channel that can stabilize membrane potentials to promote photoassimilate phloem loading and translocation. Thus, studies on functional characterization and regulatory mechanisms of AtAKT2-like channels in crops are highly important for improving crop production. Here, we identified the rice OsAKT2 as the ortholog of Arabidopsis AtAKT2, which is primarily expressed in the shoot phloem and localized at the plasma membrane. Using an electrophysiological assay, we found that OsAKT2 operated as a weakly rectifying K+ channel, preventing H+ /sucrose-symport-induced membrane depolarization. Three critical amino acid residues (K193, N206, and S326) are essential to the phosphorylation-mediated gating change of OsAKT2, consistent with the roles of the corresponding sites in AtAKT2. Disruption of OsAKT2 results in delayed growth of rice seedlings under short-day conditions. Interestingly, the lipid second messenger phosphatidic acid (PA) inhibits OsAKT2-mediated currents (both instantaneous and time-dependent components). Lipid dot-blot assay and liposome-protein binding analysis revealed that PA directly bound with two adjacent arginine residues in the ANK domain of OsAKT2, which is essential to PA-mediated inhibition of OsAKT2. Electrophysiological and phenotypic analyses also showed the PA-mediated inhibition of AtAKT2 and the negative correlation between intrinsic PA level and Arabidopsis growth, suggesting that PA may inhibit AKT2 function to affect plant growth and development. Our results functionally characterize the Shaker K+ channel OsAKT2 and reveal a direct link between phospholipid signaling and plant K+ channel modulation.

Molecular characterization of clock-associated PSEUDO-RESPONSE REGULATOR 9 gene from Oncidium ‘Gower Ramsey’

The plant circadian clock regulates a number of central activities such as growth, flowering, abiotic and biotic stress responses, and metabolism. The PSEUDO-RESPONSE REGULATORs (PRRs) contribute to the regulation of the circadian oscillator and clock output processes. In order to elucidate the role of Oncidium PRR9, we isolated and characterized a PRR9 homolog (OnPRR9) from commercially important Oncidium ‘Gower Ramsey’. OnPRR9 encodes a 603-amino acid protein with a N-terminal REC domain and a C-terminal CCT domain. Phylogenic analysis displayed that the OnPRR9 was orthologous to other known or deduced PRR proteins at the sequence level. Examination of the transcriptional expression profile of the OnPRR9 revealed that it is subjected to circadian rhythms under the conditions of 12-h light and 12-h dark cycles (12:12LD) and continuous light (LL) but not the continuous dark (DD) conditions. Genetic complementation showed that it can partially rescue the flowering time defect of Arabidopsis mutant prr5/9 grown under the long-day (16L:8D) conditions. Overexpression analysis indicated that the OnPRR9 gene can promote flowering and inhibit hypocotyl elongation under the short-day (8L:16D) conditions. Expression pattern of the OnPRR9 gene in petals during flower development and senescence implied that it may play a role in the initiation of flower senescence. These results extend our understanding of the functional conservation of PRR9 and may provide a means of regulating flowering time of Oncidium.

A suppressor of axillary meristem maturation promotes longevity in flowering plants.

Post-embryonic development and longevity of flowering plants are, for a large part, determined by the activity and maturation state of stem cell niches formed in the axils of leaves, the so-called axillary meristems (AMs)1,2. The genes that are associated with AM maturation and underlie the differences between monocarpic (reproduce once and die) annual and the longer-lived polycarpic (reproduce more than once) perennial plants are still largely unknown. Here we identify a new role for the Arabidopsis AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15) gene as a suppressor of AM maturation. Loss of AHL15 function accelerates AM maturation, whereas ectopic expression of AHL15 suppresses AM maturation and promotes longevity in monocarpic Arabidopsis and tobacco. Accordingly, in Arabidopsis grown under longevity-promoting short-day conditions, or in polycarpic Arabidopsis lyrata, expression of AHL15 is upregulated in AMs. Together, our results indicate that AHL15 and other AHL clade-A genes play an important role, directly downstream of flowering genes (SOC1, FUL) and upstream of the flowering-promoting hormone gibberellic acid, in suppressing AM maturation and extending the plant's lifespan.

Growth Responses and Flowering of <i>Kalanchoe</i> Cultivars under Elevated CO<sub>2</sub> Concentration and Varying Daylength

This experiment was conducted to identify the morphological characteristics of elevated atmospheric CO2 concentration of Kalanchoe blossfeldiana cultivars under different daylength treatments, in order to apply the carbon reduction method. CO2 treatment took place between 22:00 and 04:00 with an average of 400 μmol·mol-1 (control) and 800 μmol·mol-1 on Kalanchoe blossfeldiana ‘Lion’ and ‘Fikalrudak’. The photoperiod was controlled using an 8 h (08:00 to 16:00), 12 h (06:00 - 18:00), and 16 h (04:00 - 20:00) light-dark cycle. The number of leaves increased more in plants under the 800 μmol·mol-1 treatment than those in the control, with a photoperiod of 8 h and 12 h in both K. ‘Lion’ and 8 h in K. ‘Fikalrudak’. Plant height was lower in plants treated with 800 μmol·mol-1 CO2 than under the control treatment with an 8-h photoperiod of 8 h, regardless of K. blossfeldiana cultivars. In K. ‘Lion’, immature leaf length and width increased more in plants treated with 800 μmol·mol-1 CO2 than in the control, with a photoperiod of 8 h and 12 h. However, the leaf length and width of K. ‘Fikalrudak’ was not affected by CO2 concentration. The nodes were produced primarily by increasing CO2 concentration to 800 μmol·mol-1, regardless of daylength and K. blossfeldiana cultivars. Flowering time was not affected by daylength, however, K. ‘Lion’ bloomed within the photoperiod of 8 h and 12 h and K. ‘Fikalrudak’ bloomed only under a photoperiod of 8 h. K. blossfeldiana cultivars bloomed effectively under less light availability, regardless of CO2 concentration. These results showed that a CO2-rich enrichment promotes leaf and node production of Kalanchoe blossfeldiana cultivars under short-day conditions. Since high concentration of CO2 enrichment can have an adverse effect on flowering quality, appropriate CO2 enrichment methods for the developmental stage and cultivars should be applied on a case-by-case basis.

Alternate expression of CONSTANS-LIKE 4 in short days and CONSTANS in long days facilitates day-neutral response in Rosa chinensis.

Photoperiodic flowering responses are classified into three major types: long day (LD), short day (SD), and day neutral (DN). The inverse responses to daylength of LD and SD plants have been partly characterized in Arabidopsis and rice; however, the molecular mechanism underlying the DN response is largely unknown. Modern roses are economically important ornamental plants with continuous flowering (CF) features, and are generally regarded as DN plants. Here, RcCO and RcCOL4 were identified as floral activators up-regulated under LD and SD conditions, respectively, in the CF cultivar Rosa chinensis ‘Old-Blush’. Diminishing the expression of RcCO or/and RcCOL4 by virus-induced gene silencing (VIGS) delayed flowering time under both SDs and LDs. Interestingly, in contrast to RcCO-silenced plants, the flowering time of RcCOL4-silenced plants was more delayed under SD than under LD conditions, indicating perturbed plant responses to day neutrality. Further analyses revealed that physical interaction between RcCOL4 and RcCO facilitated binding of RcCO to the CORE motif in the promoter of RcFT and induction of RcFT. Taken together, the complementary expression of RcCO in LDs and of RcCOL4 in SDs guaranteed flowering under favorable growth conditions regardless of the photoperiod. This finding established the molecular foundation of CF in roses and further shed light on the underlying mechanisms of DN responses.

A photoperiodic time measurement served by the biphasic expression of Cryptochrome1ab in the zebrafish eye

The zebrafish (Danio rerio) is a model species that is used to study the circadian clock. It possesses light-entrainable circadian clocks in both central and peripheral tissues, and its core circadian factor cryptochromes (CRYs) have diverged significantly during evolution. In order to elucidate the functional diversity and involvement of CRYs in photoperiodic mechanisms, we investigated the daily expression profiles of six Cry transcripts in central (brain and eye) and peripheral (fin, skin and muscle) tissues. The zCry genes exhibited gene-specific diurnal conserved variations, and were divided into morning and evening groups. Notably, zCry1ab exhibited biphasic expression profiles in the eye, with peaks in the morning and evening. Comparing ocular zCry1ab expression in different photoperiods (18L:6D, 14L:10D, 10L:14D and 6L:18D) revealed that zCry1ab expression duration changed depending on the photoperiod: it increased at midnight and peaked before lights off. zCry1ab expression in constant light or dark after entrainment under long- or short-day conditions suggested that the evening clock and photic input pathway are involved in photoperiod-dependent zCry1ab expression. Laser microdissection followed by qRT-PCR analysis showed that the evening peak of zCry1ab was likely ascribed to visual photoreceptors. These results suggest the presence of an eye-specific photoperiodic time measurement served by zCry1ab.

Circumnutation and Growth of Inflorescence Stems of Arabidopsis thaliana in Response to Microgravity under Different Photoperiod Conditions.

Circumnutation is a periodic growth movement, which is an important physiological mechanism of plants to adapt to their growth environments. Gravity and photoperiod are two key environmental factors in regulating the circumnutation of plants, but the coordination mechanism between them is still unknown. In this study, the circumnutation of Arabidopsis thaliana inflorescence stems was investigated on board the Chinese recoverable satellite SJ-10 and the Chinese spacelab TG-2. Plants were cultivated in a special plant culture chamber under two photoperiod conditions [a long-day (LD) light: dark cycle of 16:8 h, and a short-day (SD) light: dark cycle of 8:16 h]. The plant growth and movements were followed by two charge-coupled device (CCD) cameras. The parameter revealed a daily (24 h) modulation on both TG-2 and SJ-10, under both the LD and the SD conditions. The inhibition of circumnutation was more apparent by microgravity under the SD in comparison with that under the LD condition, suggesting the synergistic effects of the combined microgravity and photoperiod on the circumnutation in space. In addition, an infradian rhythm (ca. 21 days long) on the TG-2 was also observed.

AtHSPR is involved in GA- and light intensity-mediated control of flowering time and seed set in Arabidopsis.

Flowering is a dynamic and synchronized process, the timing of which is finely tuned by various environmental signals. A T-DNA insertion mutant in Arabidopsis HEAT SHOCK PROTEIN-RELATED (AtHSPR) exhibited late-flowering phenotypes under both long-day (LD) and short-day (SD) conditions compared to the wild-type, while over-expression of AtHSPR promoted flowering. Exogenous application of gibberellin (GA) partially rescued the late-flowering mutant phenotype under both LD and SD conditions, suggesting that AtHSPR is involved in GA biosynthesis and/or the GA signaling that promotes flowering. Under SD or low-light conditions, the Athspr mutant exhibited late flowering together with reduced pollen viability and seed set, defective phenotypes that were partially rescued by GA treatment. qRT-PCR assays confirmed that GA biosynthetic genes were down-regulated, that GA catabolic genes were up-regulated, and that the levels of bioactive GA and its intermediates were decreased in Athspr under both SD and low-light/LD, further suggesting that AtHSPR could be involved in the GA pathway under SD and low-light conditions. Furthermore, AtHSPR interacted in vitro with OFP1 and KNAT5, which are transcriptional repressors of GA20ox1 in GA biosynthesis. Taken together, our findings demonstrate that AtHSPR plays a positive role in GA- and light intensity-mediated regulation of flowering and seed set.

Effects of day length- and temperature-regulated genes on annual transcriptome dynamics in Japanese cedar (Cryptomeria japonica D. Don), a gymnosperm indeterminate species.

Seasonal phenomena in plants are primarily affected by day length and temperature. The shoot transcriptomes of trees grown in the field and a controlled-environment chamber were compared to characterize genes that control annual rhythms and the effects of day length- and temperature-regulated genes in the gymnosperm Japanese cedar (Cryptomeria japonica D. Don), which exhibits seasonally indeterminate growth. Annual transcriptome dynamics were clearly demonstrated by principal component analysis using microarray data obtained under field-grown conditions. Analysis of microarray data from trees grown in a controlled chamber identified 2,314 targets exhibiting significantly different expression patterns under short-day (SD) and long-day conditions, and 2,045 targets exhibited significantly different expression patterns at 15°C (LT; low temperature) versus 25°C. Interestingly, although growth was suppressed under both SD and LT conditions, approximately 80% of the SD- and LT-regulated targets differed, suggesting that each factor plays a unique role in the annual cycle. The top 1,000 up-regulated targets in the growth/dormant period in the field coincided with more than 50% of the SD- and LT-regulated targets, and gene co-expression network analysis of the annual transcriptome indicated a close relationship between the SD- and LT-regulated targets. These results indicate that the respective effects of day length and temperature interact to control annual transcriptome dynamics. Well-known upstream genes of signaling pathways responsive to environmental conditions, such as the core clock (LHY/CjLHYb and CCA1/CjLHYa) and PEBP family (MFT) genes, exhibited unique expression patterns in Japanese cedar compared with previous reports in other species, suggesting that these genes control differences in seasonal regulation mechanisms between species. The results of this study provide new insights into seasonal regulation of transcription in Japanese cedar.

Characterization and fine-mapping of qHd2-1, a minor quantitative locus that affects heading date under long-day conditions in rice (Oryza sativa L.)

Heading date, a complex quantitative trait, determines regional adaptability and is associated with grain yield and quality in rice. In this study, we characterized qHd2-1, a minor-effect quantitative locus (QTL) for promoting heading date (by 3–4 days) under long-day (LD) conditions, but not short-day (SD) conditions. The qHd2-1 locus was detected in single segment substitution line (SSSL) N22-1, which was developed using indica 9311 as the donor and japonica Nipponbare (NP) as the recipient. A set of substitution lines were developed from a segregating population derived from a cross between N22-1 and NP. Based on the genotypic identification and phenotypic evaluation of the target substitution lines, qHd2-1 was narrowed down to a 105-kb region between STS2-20 and STS2-22 on the short arm of chromosome 2. Furthermore, qHd2-1 was found to function by upregulating the florigen genes Hd3a and RFT1 during floral induction under LD conditions. Field tests showed that qHd2-1 promotion of heading date affects grain yield‑related agronomic traits, such as plant height, panicle number per plant, and flag leaf size, as well as thousand-grain weight and grain width. Taken together, these findings establish a foundation for cloning this minor-effect QTL involved in heading date, and qHd2-1 could be of great significance in rice breeding to allow the fine-tuning of heading date in rice cultivars.

Effect of postnatal photoperiod on DNA methylation dynamics in the mouse brain

Season of birth influences the onset of psychiatric diseases in mammals. Recent studies using rodent models have revealed that photoperiod during early life stages has a strong impact on affective and cognitive behaviors, neuronal activity, and hippocampal neurogenesis/astrogenesis in later life. The present study examined the effect of postnatal photoperiod on global DNA methylation and hydroxymethylation dynamics in the mouse brain. Male mice born under short-day (SD) conditions were divided into SD and long-day (LD) groups on the day of birth. Temporal expression of DNA methyltransferases (DNMT1/3a) with 5-methylcytosine (5-mC) levels, as well as protein levels of ten-eleven translocation (TET) 2 with 5-hydroxymethylcytosine (5-hmC) levels, were analyzed from postnatal day 4 (P4) to P21. Levels of 5-hmC in all hippocampal areas were higher in the LD group than in the SD group at P21, with a positive correlation between 5-hmC levels and TET2 levels throughout the experimental period. Inconsistent results were observed between DNMT1/3a mRNA levels and 5-mC levels. On the other hand, in the OB, mRNA levels of DNMT1 and DNMT3a were slightly lower in the LD group similar to 5-mC levels, but TET2 and 5-hmC levels were not influenced by the photoperiod. In conclusion, postnatal exposure of mice to LD conditions induces an increase in TET2-dependent DNA hydroxymethylation in the hippocampus, which might be involved in the long-term effects of postnatal photoperiod on neurogenesis and affective/cognitive behaviors.

Genome-wide identification of cold responsive transcription factors in Brassica napus L

BackgroundCold stress is one of the primary environmental factors that affect plant growth and productivity, especially for crops like Brassica napus that live through cold seasons. Till recently, although a number of genes and pathways involved in B. napus cold response have been revealed by independent studies, a genome-wide identification of the key regulators and the regulatory networks is still lack. In this study, we investigated the transcriptomes of cold stressed semi-winter and winter type rapeseeds in short day condition, mainly with the purpose to systematically identify the functional conserved transcription factors (TFs) in cold response of B. napus.ResultsGlobal modulation of gene expression was observed in both the semi-winter type line (158A) and the winter type line (SGDH284) rapeseeds, in response to a seven-day chilling stress in short-day condition. Function analysis of differentially expressed genes (DEGs) revealed enhanced stresses response mechanisms and inhibited photosynthesis in both lines, as well as a more extensive inhibition of some primary biological processes in the semi-winter type line. Over 400 TFs were differentially expressed in response to cold stress, including 56 of them showed high similarity to the known cold response TFs and were consistently regulated in 158A and SGDH284, as well as 25 TFs which targets were over-represented in the total DEGs. A further investigation based on their interactions indicated the critical roles of several TFs in cold response of B. napus.ConclusionIn summary, our results revealed the alteration of gene expression in cold stressed semi-winter and winter ecotype B. napus lines and provided a valuable collection of candidate key regulators involved in B. napus response to cold stress, which could expand our understanding of plant stress response and benefit the future improvement of the breed of rapeseeds.

Minor Effects of 11 Dof Family Genes Contribute to the Missing Heritability of Heading Date in Rice (Oryza sativa L.).

DNA binding with one finger (Dof) proteins are plant-specific transcription factors with important and diverse functions in seed germination, flowering time, and biotic and abiotic stresses. In this study, haplotype-based association analysis was conducted between heading date and 30 Dof family genes in a worldwide germplasm collection. Of these, 22 Dof genes were associated with heading date. Multiple comparisons among haplotypes revealed their diverse functions in promoting and suppressing heading date under short-day (SD) and long-day (LD) conditions. They cumulatively made a considerable contribution to the missing heritability of heading date. A set of knockout mutants of 30 Dof genes generated by CRISPR/Cas9-mediated genome editing technology showed that 11 and 9 Dof genes regulated heading date under LD and SD, respectively. Phenotype measurement of mutants showed that these 11 and 9 Dof genes slightly regulated heading with effects of 2–5 days under LD and SD, respectively. Both mutant and natural variation assays indicated functional redundancy in regulating heading date among Dof family genes. Nucleotide diversity analysis suggested that most Dof genes have been subjected to selection during domestication and improvement. Beyond heading date, this set of mutants is also a good resource for evaluating the function of Dof genes in regulating stress tolerance and seed germination.

Seasonal phenotype-specific expression of microRNAs during metamorphosis in the European map butterfly Araschnia levana.

The European map butterfly (Araschnia levana) is a well-known example of seasonal polyphenism because the spring and summer imagoes exhibit distinct morphological phenotypes. The day length and temperature during larval and prepupal development determine whether spring or summer imagoes emerge after metamorphosis. Inspired by the fundamentally different transcriptomic profiles in prepupae developing from larvae exposed to long days or short days, we postulate that posttranscriptional epigenetic regulators such as microRNAs (miRNAs) may contribute to the epigenetic control of seasonal polyphenism in A. levana. To test this hypothesis, we used microarrays containing over 2,000 insect miRNAs to identify candidate regulators that are differentially expressed in last-instar larvae or pupae developing under long-day or short-day conditions. We used our transcriptomic database to identify potential 3'-untranslated regions of messenger RNAs to predict miRNA targets by considering both base pair complementarity and minimum free energy hybridization. This approach resulted in the identification of multiple targets of miRNAs that were differentially regulated in polyphenic morphs of A. levana including a candidate (miR-2856-3p) regulating the previously identified diapause bioclock protein gene. In conclusion, the expression profiling of miRNAs provided insights into their possible involvement in seasonal polyphenism of A. levana and offer an important resource for further studies.