Trojan Horse Strategy for Non-invasive Interference of <Emphasis Type="Italic">Clock</Emphasis> Gene in the Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

Substrates of the chloroplast small heat shock proteins 22E/F point to thermolability as a regulative switch for heat acclimation in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

We previously described a Brassica napus chlorophyll-deficient mutant (ygl) with yellow-green seedling leaves and mapped the related gene, BnaC.YGL, to a 0.35 cM region. However, the molecular mechanisms involved in this chlorophyll defect are still unknown. In this study, the BnaC07.HO1 gene (equivalent to BnaC.YGL) was isolated by the candidate gene approach, and its function was confirmed by genetic complementation. Comparative sequencing analysis suggested that BnaC07.HO1 was lost in the mutant, while a long noncoding-RNA was inserted into the promoter of the homologous gene BnaA07.HO1. This insert was widely present in B. napus cultivars and down-regulated BnaA07.HO1 expression. BnaC07.HO1 was highly expressed in the seedling leaves and encoded heme oxygenase 1, which was localized in the chloroplast. Biochemical analysis showed that BnaC07.HO1 can catalyze heme conversion to form biliverdin IXα. RNA-seq analysis revealed that the loss of BnaC07.HO1 impaired tetrapyrrole metabolism, especially chlorophyll biosynthesis. According, the levels of chlorophyll intermediates were reduced in the ygl mutant. In addition, gene expression in multiple pathways was affected in ygl. These findings provide molecular evidences for the basis of the yellow-green leaf phenotype and further insights into the crucial role of HO1 in B. napus.     

PbCOL8 is a clock-regulated flowering time repressor in pear

The floral transition is controlled by diverse endogenous and exogenous cues. In many species, COL (CONSTANS-like) genes integrate light and circadian clock signals to regulate flowering time. However, little is known about COLs in perennial woody plants. Here, we identified 15 PbCOLs in pear (Pyrus bretschneideri). PbCOLs were classified into three groups by phylogenetic tree analysis using protein sequences. Multiple sequence alignment analysis revealed conserved B-box and CCT (CO, CO-like, and TOC1) domains in all PbCOL members. This result suggested that PbCOLs might possess conserved functions as other species. Six PbCOLs were found to be regulated by both circadian clock and photoperiod. Here, we showed that PbCOL8, a member of group 2, suppressed the flowering signal integrators FT and SOC1 and could repress flowering time. These findings will contribute to elucidation of the mechanism of floral initiation in pear.     

Overexpression and loss-of-function at <Emphasis Type="Italic">TIME FOR COFFEE</Emphasis> results in similar phenotypes in diverse growth and physiological responses

Plant experience diurnal changes in their environment that can be anticipated and responded to via the circadian clock. Integration of external signals by this clock ensures metabolic homeostasis and ultimately enhances fitness. TIME FOR COFFEE (TIC) is known to be associated to the circadian clock, being required to maintain rhythmic period and amplitude, and to regulate clock-driven physiological responses. The molecular function of TIC has so far only been studied with loss-of-function mutants. The biochemical activity of TIC remains elusive. To learn more about TIC in diverse physiological processes, here we generated TIC overexpressing plants (TICox) and characterized their impact on plant growth, development, and circadianclock activity. TICox plants displayed phenotypic similarity with tic mutants. This included defects in leaf morphology, the developmental transition from the vegetative to reproductive phase, and circadian-clock function. These observations allowed us to hypothesize that TIC is an element of protein complexes that are involved in global biological processes.     

Phenotypic effects of genetic variability in human clock genes on circadian and sleep parameters

Circadian rhythms and sleep are two separate but intimately related processes. Circadian rhythms are generated through the precisely controlled, cyclic expression of a number of genes designated clock genes. Genetic variability in these genes has been associated with a number of phenotypic differences in circadian as well as sleep parameters, both in mouse models and in humans. Diurnal preferences as determined by the selfreported Horne-Östberg (HÖ) questionnaire, has been associated with polymorphisms in the human genes CLOCK, PER1, PER2 and PER3. Circadian rhythm-related sleep disorders have also been associated with mutations and polymorphisms in clock genes, with the advanced type cosegrating in an autosomal dominant inheritance pattern with mutations in the genes PER2 and CSNK1D, and the delayed type associating without discernible Mendelian inheritance with polymorphisms in CLOCK and PER3. Several mouse models of clock gene null alleles have been demonstrated to have affected sleep homeostasis. Recent findings have shown that the variable number tandem polymorphism in PER3, previously linked to diurnal preference, has profound effects on sleep homeostasis and cognitive performance following sleep loss, confirming the close association between the processes of circadian rhythms and sleep at the genetic level.     

Involvement of Nuclear Receptor REV-ERBβ in Formation of Neurites and Proliferation of Cultured Adult Neural Stem Cells

Neural stem cells (NSCs) serve as the source of both neurons and support cells, and neurogenesis is reportedly linked to the circadian clock. This study aimed to clarify the functional role of the circadian rhythm-related nuclear receptor, REV-ERBβ, in neurogenesis of NSCs from adult brain. Accordingly, Rev-erbβ expression and the effect of Rev-erbβ gene-specific knockdown on neurogenesis in vitro was examined in adult rodent NSCs. Initial experiments confirmed REV-ERBβ expression in cultured adult NSCs, while subsequent gene expression and gene ontogeny analyses identified functional genes upregulated or downregulated by REV-ERBβ. In particular, expression levels of factors associated with proliferation, stemness, and neural differentiation were affected. Knockdown of Rev-erbβ showed involvement of REV-ERBβ in regulation of cellular proliferation and self-renewal of cultured adult NSCs. Moreover, Rev-erbβ-knockdown cells formed neurons with a slightly shrunken morphology, fewer new primary neurites, and reduced length and branch formation of neurites. Altogether, this suggests that REV-ERBβ is involved in neurite formation during neuronal differentiation of cultured adult NSCs. In summary, REV-ERBβ is a known circadian regulatory protein that appears to be involved in neurogenesis via regulation of networks for cell proliferation and neural differentiation/maturation in adult NSCs.     

Localization of the <Emphasis Type="Italic">PITX2</Emphasis> gene expression in human eye cells in the course of prenatal development

The pattern of the PITX2 gene expression was studied in the cornea, lens, retina, iridocorneal complex (ICC), and eye coats of human fetuses at weeks 9.5–22 of intrauterine development. Using the PCR method, PITX2 expression in all these tissues was revealed already at the earliest stage studied (9.5 weeks), being especially strong in the anterior eye complex (the cornea and lens) and weaker in the retina and sclera. The level of PITX2 expression in all eye tissues slightly decreased by week 15, increased to a high level in the ICC on week 18, and further decreased in all tissues by week 22. Using cDNA derived from the whole eyes of 8-, 9-, 10.5-, and 11-week fetuses, the expression of two PITX2 isoforms specific for eye tissues (A and B) was revealed. By means of in situ hybridization, the PITX2 mRNA was localized in the eye tissues of ectodermal and neuroectodermal origin.     

The alternative splicing of <Emphasis Type="Italic">EAM8</Emphasis> contributes to early flowering and short-season adaptation in a landrace barley from the Qinghai-Tibetan Plateau

Key message

The early flowering of Lalu was determined to be due to a novel spontaneous eam8 mutation, which resulted in intron retention and the formation of a putative truncated protein.

Abstract

Barley is a staple crop grown over an extensive area in the Qinghai-Tibetan Plateau. Understanding the genetic mechanism for its success in a high altitude is important for crop improvement in marginal environments. Early flowering is a critical adaptive trait that strongly influences reproductive fitness in a short growing season. Loss-of-function mutations at the circadian clock gene EARLY MATURITY 8 (EAM8) promote rapid flowering. In this study, we identified a novel, spontaneous recessive eam8 mutant with an early flowering phenotype in a Tibetan barley landrace Lalu, which is natively grown at a high altitude of approximately 4000 m asl. The co-segregation analysis in a F₂ population derived from the cross Lalu (early flowering)?×?Diqing 1 (late flowering) confirmed that early flowering of Lalu was determined to be due to an allele at EAM8. The eam8 allele from Lalu carries an A/G alternative splicing mutation at position 3257 in intron 3, designated eam8.l; this alternative splicing event leads to intron retention and a putative truncated protein. Of the 134 sequenced barley accessions, which are primarily native to the Qinghai-Tibet Plateau, three accessions carried this mutation. The eam8.l mutation was likely to have originated in wild barley due to the presence of the Lalu haplotype in H. spontaneum from Tibet. Overall, alternative splicing has contributed to the evolution of the barley circadian clock and in the short-season adaptation of local barley germplasm. The study has also identified a novel donor of early-flowering barley which will be useful for barley improvement.

     

Expression of Vsx transcription factors in the morphogenesis of retina in the chicken <Emphasis Type="Italic">Gallus domesticus</Emphasis>

Using PCR analysis and immunofluorescence staining, we have investigated the expression of homeobox genes Vsx1/Chx10-1 and Vsx2/Chx10 from the Vsx family (visual system homeobox) during retinal morphogenesis in the chicken Gallus domesticus. It was found that the expression of the studied genes starts at the early stages of embryogenesis. It was shown that the proteins of Vsx1 and Vsx2 are localized in the bipolar cells of the inner nuclear layer of the forming retina. The participation of Vsx1/Chx10-1 and Vsx2/Chx1 in the regulation of retinal cell differentiation in various species of vertebrates and in humans was discussed.