AabHLH48, a novel basic helix-loop-helix transcription factor from Adonis amurensis, promotes early flowering in Arabidopsis by activating FRUITFULL expression.

Basic helix-loop-helix (bHLH) transcription factors play various important roles in plant growth and development. In this study, a AabHLH48 was identified in the floral organ of Adonis amurensis, a perennial herb that can naturally complete flowering at extreme low temperatures. AabHLH48 was widely expressed in various tissues or organs of A. amurensis and was localized in the nucleus. Overexpression of AabHLH48 promotes early flowering in Arabidopsis under both photoperiod (12 h light/12 h dark and 16 h light/8 h dark) and temperature (22 and 18 °C) conditions. Transcriptome sequencing combined with quantitative real-time PCR analysis showed that overexpression of AabHLH48 caused a general upregulation of genes involved in floral development in Arabidopsis, especially for AtAGAMOUS-LIKE 8/FRUITFULL (AtAGL8/FUL). The yeast one-hybrid assay revealed that AabHLH48 has transcriptional activating activity and can directly bind to the promoter region of AtAGL8/FUL. These results suggest that the overexpression of AabHLH48 promoting early flowering in Arabidopsis is associated with the upregulated expression of AtAGL8/FUL activated by AabHLH48. This indicates that AabHLH48 can serve as an important genetic resource for improving flowering-time control in other ornamental plants or crops.

Dynamic transcriptome analysis provides molecular insights into underground floral differentiation in Adonis Amurensis Regel & Radde.

Understanding flower developmental processes is a prerequisite for improving flowering 'plants' production. Adonis amurensis is a fascinating spring ephemeral plant that develops its flower organs underground. Nevertheless, knowledge of the molecular mechanisms driving this particular process is scarce. Herein, we examined transcriptional changes during underground flower differentiation in A. amurensis and unveiled key differently regulated genes and pathways. High-throughput RNA sequencing of meristems at different flower developmental stages, including flower primordium (FP), sepal stage (SE), perianth primordium (PE), stamen stage (ST), and pistil stage (PI), identified 303,234 unigenes that showed 44.79% similarity with sequences in Aquilegia coerulea. Correlations, principal component, and differentially expressed genes (DEGs) analyses revealed that few molecular changes occurred during the transition from PE to ST. Many DEGs exhibited stage-specific regulations. Transcription factor (TF) and phytohormone family genes are critical regulators of the floral differentiation process in A. amurensis. The most differentially regulated TFs were MADS, FAR1, MYBs, AP2/ERF, B3, C2H2, and LOBs. We filtered out 186 candidate genes for future functional studies, including 18 flowering/circadian-related, 32 phytohormone-related, and TF family genes. Our findings deepen our understanding of the underground flower differentiation process and offer critical resources to dissect its regulatory network in A. amurensis. These findings establish a foundational platform for researchers dedicated to exploring the unique phenotypic characteristics of this specific flowering modality and delving into the intricate molecular mechanisms underpinning its regulation and expression.

Identification of Transcription Factors Involved in the Regulation of Flowering in Adonis Amurensis Through Combined RNA-seq Transcriptomics and iTRAQ Proteomics.

Temperature is one of the most important environmental factors affecting flowering in plants. Adonis amurensis, a perennial herbaceous flower that blooms in early spring in northeast China where the temperature can drop to -15 °C, is an ideal model for studying the molecular mechanisms of flowering at extremely low temperatures. This study first investigated global gene expression profiles at different developmental stages of flowering in A. amurensis by RNA-seq transcriptome and iTRAQ proteomics. Finally, 123 transcription factors (TFs) were detected in both the transcriptome and the proteome. Of these, 66 TFs belonging to 14 families may play a key role in multiple signaling pathways of flowering in A. amurensis. The TFs FAR1, PHD, and B3 may be involved in responses to light and temperature, while SCL, SWI/SNF, ARF, and ERF may be involved in the regulation of hormone balance. SPL may regulate the age pathway. Some members of the TCP, ZFP, MYB, WRKY, and bHLH families may be involved in the transcriptional regulation of flowering genes. The MADS-box TFs are the key regulators of flowering in A. amurensis. Our results provide a direction for understanding the molecular mechanisms of flowering in A. amurensis at low temperatures.

The AaDREB1 Transcription Factor from the Cold-Tolerant Plant Adonis amurensis Enhances Abiotic Stress Tolerance in Transgenic Plant.

Dehydration-responsive element binding (DREB) transcription factors (TFs) play important roles in the regulation of plant resistance to environmental stresses and can specifically bind to dehydration-responsive element/C-repeat element (DRE/CRT) proteins (G/ACCGAC) and activate expression of many stress-inducible genes. Here, we cloned and characterized a novel gene (AaDREB1) encoding the DREB1 transcription factor from the cold-tolerant plant Adonis amurensis. Quantitative real-time (qRT)-PCR results indicated that AaDREB1 expression was induced by salt, drought, cold stress, and abscisic acid application. A yeast one-hybrid assay demonstrated that AaDREB1 encodes a transcription activator and specifically binds to DRE/CRT. Furthermore, transgenic Arabidopsis and rice harboring AaDREB1 showed enhanced tolerance to salt, drought, and low temperature. These results indicated that AaDREB1 might be useful in genetic engineering to improve plant stress tolerance.

Elucidation of the pathway to astaxanthin in the flowers of Adonis aestivalis.

A few species in the genus Adonis are the only land plants known to produce the valuable red ketocarotenoid astaxanthin in abundance. Here, we ascertain the pathway that leads from the ß-rings of ß-carotene, a carotenoid ubiquitous in plants, to the 3-hydroxy-4-keto-ß-rings of astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione) in the blood-red flowers of Adonis aestivalis, an ornamental and medicinal plant commonly known as summer pheasant's eye. Two gene products were found to catalyze three distinct reactions, with the first and third reactions of the pathway catalyzed by the same enzyme. The pathway commences with the activation of the number 4 carbon of a ß-ring in a reaction catalyzed by a carotenoid ß-ring 4-dehydrogenase (CBFD), continues with the further dehydrogenation of this carbon to yield a carbonyl in a reaction catalyzed by a carotenoid 4-hydroxy-ß-ring 4-dehydrogenase, and concludes with the addition of an hydroxyl group at the number 3 carbon in a reaction catalyzed by the erstwhile CBFD enzyme. The A. aestivalis pathway is both portable and robust, functioning efficiently in a simple bacterial host. Our elucidation of the pathway to astaxanthin in A. aestivalis provides enabling technology for development of a biological production process and reveals the evolutionary origin of this unusual plant pathway, one unrelated to and distinctly different from those used by bacteria, green algae, and fungi to synthesize astaxanthin.

Development of an Adonis aestivalis expressed sequence tag population as a resource for genes of the carotenoid pathway.

Expressed sequence tag (EST) libraries are a powerful tool for gene discovery in plants with genomes that are not fully sequenced. Adonis aestivalis (Ranunculaceae) is unusual among higher plants in that it accumulates large amounts of the valuable red ketocarotenoid astaxanthin in the flower petals, in addition to other carotenoids, and should therefore be a rich source of transcripts involved in carotenoid biosynthesis. To create a resource for gene discovery and increase our understanding of ketocarotenoid biosynthesis in plants, we sequenced 4189 cloned cDNAs from the floral tissues of A. aestivalis. The EST collection was annotated using sequence homology searches comparing the A. aestivalis ESTs with reference databases. ESTs involved in carotenoid biosynthesis were identified based on sequence homology to published sequences and by functional prediction using InterProScan. This study identified expressed transcripts for most of the known genes involved in the carotenoid pathway. Using an Escherichia coli functional expression system, two candidate transcripts from the A. aestivalis cDNA library were functionally characterized and identified as a novel beta-carotene 3-hydroxylase (CrtH2) and a beta-carotene monooxygenase. The expanded use of this EST collection combined with the E. coli expression system as presented here will greatly facilitate the cloning and characterization of candidate genes involved in carotenoid biosynthesis in plants.

Cost of reproduction in a spring ephemeral species, Adonis ramosa (Ranunculaceae): carbon budget for seed production.

BACKGROUND AND AIMS: Spring ephemerals have a specific life-history trait, i.e. shoot growth and sexual reproduction occur simultaneously during a short period from snowmelt to canopy closure in deciduous forests. The aim of this study is to clarify how spring ephemerals invest resources for seed production within a restricted period. METHODS: In order to evaluate the cost of reproduction of a typical spring ephemeral species, Adonis ramosa, an experiment was conducted comprising defoliation treatments (intact, one-third and two-thirds leaf-cutting) and fruit manipulations (control, shading and removal) over two growing seasons. In addition, measurements were made of the movements of carbon assimilated via (13)C tracing. KEY RESULTS: Survival rate was high irrespective of treatments and manipulations. The proportion of flowering plants and plant size decreased as a result of the defoliation treatments over 2 years, but the fruit manipulations did not affect flowering activity or plant size. Seed set and seed number decreased as a result of fruit shading treatment, but the defoliation treatments did not affect current seed production. Individual seed weight also decreased in the second year due to fruit shading. The (13)C tracing experiment revealed that young fruits had photosynthetic ability and current photosynthetic products from leaves were mainly transferred to the below-ground parts, while translocation to fruit was very small even when fruit photosynthesis was restricted by the shading treatment. CONCLUSIONS: Current foliage photosynthetic products are largely stored in the below-ground parts for survival and future growth, and about one-third of the resources for seed production may be attained by fruit photosynthesis. Therefore, the trade-off between current seed production and subsequent growth is weak. The cost of seed production may be buffered by sufficient storage in the below-ground organs, effective photosynthesis under high irradiation and self-assimilation ability of fruits.

A study in scarlet: enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis.

The red ketocarotenoid astaxanthin (3,3'-dihydroxy-4,4'-diketo-beta,beta-carotene) is widely used as an additive in feed for the pigmentation of fish and crustaceans and is frequently included in human nutritional supplements as well. There is considerable interest in developing a plant-based biological production process for this valuable carotenoid. Adonis aestivalis (Ranunculaceae) is unusual among plants in synthesizing and accumulating large amounts of astaxanthin and other ketocarotenoids. The formation of astaxanthin requires only the addition of a carbonyl at the number 4 carbon of each beta-ring of zeaxanthin (3,3'-dihydroxy-beta,beta-carotene), a carotenoid typically present in the green tissues of higher plants. We screened an A. aestivalis flower library to identify cDNAs that might encode the enzyme that catalyzes the addition of the carbonyls. Two closely related cDNAs selected in this screen were found to specify polypeptides similar in sequence to plant beta-carotene 3-hydroxylases, enzymes that convert beta-carotene (beta,beta-carotene) into zeaxanthin. The Adonis enzymes, however, exhibited neither 4-ketolase nor 3-hydroxylase activity when presented with beta-carotene as the substrate in Escherichia coli. Instead, the products of the Adonis cDNAs were found to modify beta-rings in two distinctly different ways: desaturation at the 3,4 position and hydroxylation of the number 4 carbon. The 4-hydroxylated carotenoids formed in E. coli were slowly metabolized to yield compounds with ketocarotenoid-like absorption spectra. It is proposed that a 3,4-desaturation subsequent to 4-hydroxylation of the beta-ring leads to the formation of a 4-keto-beta-ring via an indirect and unexpected route: a keto-enol tautomerization.