Whirly Family Research Articles

Genome-Wide Identification and Molecular Evolutionary History of the Whirly Family Genes in Brassica napus.

Whirly transcription factors are unique to plants, playing pivotal roles in managing leaf senescence and DNA repair. While present in various species, their identification in Brassica napus L. (B. napus) and their differences during hybridization and polyploidy has been elusive. Addressing this, our study delves into the functional and evolutionary aspects of the Whirly gene family during the emergence of B. napus, applying bioinformatics and comparative genomics. We identified six Whirly genes in B. napus. In Brassica rapa L. (B. rapa), three Whirly genes were identified, while four were found in Brassica oleracea L. (B. oleracea). The results show that the identified Whirly genes not only have homology but also share the same chromosomal positions. Phylogenetic analysis indicates that Whirly genes in monocots and dicots exhibit high conservation. In the evolutionary process, the Whirly gene family in B. napus experienced events of intron/exon loss. Collinearity insights point to intense purifying selection post-duplication. Promoter regions housed diverse cis-acting elements linked to photoresponse, anaerobic initiation, and methyl jasmonate responsiveness. Notably, elements tied to abscisic acid signaling and meristem expression were prominent in diploid ancestors but subdued in tetraploid B. napus. Tissue-specific expression unveiled analogous patterns within subfamily genes. Subsequent qRT-PCR analysis spotlighted BnAWHY1b's potential significance in abiotic stress response, particularly drought. These findings can be used as theoretical foundations to understand the functions and effects of the Whirly gene family in B. napus, providing references for the molecular mechanism of gene evolution between this species and its diploid ancestors.

Genome-wide identification, phylogenetic, and expression analysis under abiotic stress conditions of Whirly (WHY) gene family in Medicago sativa L.

The WHY family is a group of plant-specific transcription factors, that can bind to single-stranded DNA molecules and play a variety of functions in plant nuclei and organelles, participating in the regulation of plant leaf senescence. It has been identified and analyzed in many species, however, the systematic identification and analysis of the WHY genes family have not yet been reported in alfalfa (Medicago sativa L.). Therefore, to explore the function of alfalfa the WHY genes, and 10 MsWHY genes were identified and further characterized their evolutionary relationship and expression patterns by analyzing the recently published genome of alfalfa. Comprehensive analysis of the chromosome location, physicochemical properties of the protein, evolutionary relationship, conserved motifs, and responses to abiotic stresses of the WHY gene family in alfalfa using bioinformatics methods. The results showed that 10 MsWHY genes were distributed on 10 chromosomes, and collinearity analysis showed that many MsWHYs might be derived from segmental duplications, and these genes are under purifying selection. Based on phylogenetic analyses, the WHY gene family of alfalfa can be divided into four subfamilies: I-IV subfamily, and approximately all the WHY genes within the same subfamily share similar gene structures. The 10 MsWHY gene family members contained 10 motifs, of which motif 2 and motif 4 are the conserved motifs shared by these genes. Furthermore, the analysis of cis-regulatory elements indicated that regulatory elements related to transcription, cell cycle, development, hormone, and stress response are abundant in the promoter sequence of the MsWHY genes. Real-time quantitative PCR demonstrated that MsWHYs gene expression is induced by drought, salt, and methyl jasmonate. The present study serves as a basic foundation for future functional studies on the alfalfa WHY family.

An Insight Into the Mechanism of Plant Organelle Genome Maintenance and Implications of Organelle Genome in Crop Improvement: An Update.

Besides the nuclear genome, plants possess two small extra chromosomal genomes in mitochondria and chloroplast, respectively, which contribute a small fraction of the organelles’ proteome. Both mitochondrial and chloroplast DNA have originated endosymbiotically and most of their prokaryotic genes were either lost or transferred to the nuclear genome through endosymbiotic gene transfer during the course of evolution. Due to their immobile nature, plant nuclear and organellar genomes face continuous threat from diverse exogenous agents as well as some reactive by-products or intermediates released from various endogenous metabolic pathways. These factors eventually affect the overall plant growth and development and finally productivity. The detailed mechanism of DNA damage response and repair following accumulation of various forms of DNA lesions, including single and double-strand breaks (SSBs and DSBs) have been well documented for the nuclear genome and now it has been extended to the organelles also. Recently, it has been shown that both mitochondria and chloroplast possess a counterpart of most of the nuclear DNA damage repair pathways and share remarkable similarities with different damage repair proteins present in the nucleus. Among various repair pathways, homologous recombination (HR) is crucial for the repair as well as the evolution of organellar genomes. Along with the repair pathways, various other factors, such as the MSH1 and WHIRLY family proteins, WHY1, WHY2, and WHY3 are also known to be involved in maintaining low mutation rates and structural integrity of mitochondrial and chloroplast genome. SOG1, the central regulator in DNA damage response in plants, has also been found to mediate endoreduplication and cell-cycle progression through chloroplast to nucleus retrograde signaling in response to chloroplast genome instability. Various proteins associated with the maintenance of genome stability are targeted to both nuclear and organellar compartments, establishing communication between organelles as well as organelles and nucleus. Therefore, understanding the mechanism of DNA damage repair and inter compartmental crosstalk mechanism in various sub-cellular organelles following induction of DNA damage and identification of key components of such signaling cascades may eventually be translated into strategies for crop improvement under abiotic and genotoxic stress conditions. This review mainly highlights the current understanding as well as the importance of different aspects of organelle genome maintenance mechanisms in higher plants.

Identifying strawberry Whirly family transcription factors and their expressions in response to crown rot

Crown rot is one of the most destructive diseases of cultivated strawberry. The correlation between Whirly family transcription factors, the one class of known resistance genes, and strawberry crown rot resistance has not been studied. In this study, the Whirlys of Fragaria × ananassa, F. iinumae, F. vesca, F. viridis and F. nilgerrensis were characterized by searching the strawberry genome database and analyzing the presence of Whirly domains. Five FaWHYs, two FiWHYs, three FnWHYs, two FviWHYs and four FvWHYs were identified from their respective genome. Two gene clusters with segmental duplications were obtained from the gene cluster analysis with two and three FaWHYs, and three FaWHYs showed syntenic relationships with AtWHYs of Arabidopsis thaliana. FiWHY1, FvWHY2 and FviWHY1 showed syntenic relationships with FaWHY1 and FaWHY2. At the same time, FiWHY2, FvWHY3, FviWHY2 and FnWHY3 exhibited similar syntenic relationships with FaWHY4 and FaWHY5. In addition, FnWHY1 and FnWHY2 corresponded to both FaWHY1 and FaWHY2. Gene expression analysis revealed that five FaWHYs were expressed in crowns, and the regulation of FaWHYs was always consistent with the cis-elements in their promoters. All of them were downregulated by crown rot infected. Together, these results provided a basis for further functional studies of the FaWHYs proteins and their responses to crown rot.

Overexpression of tomato WHIRLY protein enhances tolerance to drought stress and resistance to Pseudomonas solanacearum in transgenic tobacco

WHIRLY transcription factors play critical roles in responses to biotic and abiotic stresses, but their other biological functions remain unclear. In this study, SlWHY2, a member of the WHIRLY family, was isolated from Solanum lycopersicum. The role of SlWHY2 was studied using transgenic tobacco plants. Real-time quantitative polymerase chain reaction analysis showed that SlWHY2 expression was induced by polyethylene glycol, NaCl, salicylic acid, hydrogen peroxide, and bacterial pathogens. SlWHY2 overexpression in tobacco caused enhanced tolerance to drought stress, as indicated by lower accumulation of malondialdehyde and relative electrolyte leakage, as well as higher relative water content and activities of superoxide dismutase and ascorbate peroxidase. Moreover, higher expression of cytochrome oxidase 1 (NtCOX1) and open reading frame 1 (NtORF1) were observed under drought in the transgenic lines. This suggested that overexpression of SlWHY2 enhanced tolerance to drought stress by regulating the transcription of mitochondrial genes and stabilizing mitochondrial function. Transgenic tobacco also displayed greater resistance to Pseudomonas solanacearum infection as evidenced by lower reactive oxygen species content and higher expression of defence-related genes. Overall, these findings suggest that SlWHY2 acts as a positive regulator in response to biotic and abiotic stresses.

The structure of a contact-dependent growth-inhibition (CDI) immunity protein from Neisseria meningitidis MC58.

Contact-dependent growth inhibition (CDI) is an important mechanism of intercellular competition between neighboring Gram-negative bacteria. CDI systems encode large surface-exposed CdiA effector proteins that carry a variety of C-terminal toxin domains (CdiA-CTs). All CDI(+) bacteria also produce CdiI immunity proteins that specifically bind to the cognate CdiA-CT and neutralize its toxin activity to prevent auto-inhibition. Here, the X-ray crystal structure of a CdiI immunity protein from Neisseria meningitidis MC58 is presented at 1.45 Å resolution. The CdiI protein has structural homology to the Whirly family of RNA-binding proteins, but appears to lack the characteristic nucleic acid-binding motif of this family. Sequence homology suggests that the cognate CdiA-CT is related to the eukaryotic EndoU family of RNA-processing enzymes. A homology model is presented of the CdiA-CT based on the structure of the XendoU nuclease from Xenopus laevis. Molecular-docking simulations predict that the CdiA-CT toxin active site is occluded upon binding to the CdiI immunity protein. Together, these observations suggest that the immunity protein neutralizes toxin activity by preventing access to RNA substrates.

The functions of WHIRLY1 and REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 in cross tolerance responses in plants: a hypothesis

Chloroplasts are important sensors of environment change, fulfilling key roles in the regulation of plant growth and development in relation to environmental cues. Photosynthesis produces a repertoire of reductive and oxidative (redox) signals that provide information to the nucleus facilitating appropriate acclimation to a changing light environment. Redox signals are also recognized by the cellular innate immune system allowing activation of non-specific, stress-responsive pathways that underpin cross tolerance to biotic-abiotic stresses. While these pathways have been intensively studied in recent years, little is known about the different components that mediate chloroplast-to-nucleus signalling and facilitate cross tolerance phenomena. Here, we consider the properties of the WHIRLY family of proteins and the REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 (RRTF1) in relation to chloroplast redox signals that facilitate the synergistic co-activation of gene expression pathways and confer cross tolerance to abiotic and biotic stresses. We propose a new hypothesis for the role of WHIRLY1 as a redox sensor in chloroplast-to-nucleus retrograde signalling leading to cross tolerance, including acclimation and immunity responses. By virtue of its association with chloroplast nucleoids and with nuclear DNA, WHIRLY1 is an attractive candidate coordinator of the expression of photosynthetic genes in the nucleus and chloroplasts. We propose that the redox state of the photosynthetic electron transport chain triggers the movement of WHIRLY1 from the chloroplasts to the nucleus, and draw parallels with the regulation of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1).

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA-binding protein ODB1 was identified from a mitochondrial extract after DNA-affinity purification. ODB1 (organellar DNA-binding protein 1) co-purified with WHY2, a mitochondrial member of the WHIRLY family of plant-specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52-1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo-protein complexes. Both proteins co-immunoprecipitated in a DNA-dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single-stranded than for double-stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co-immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52-like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild-type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology-mediated recombination. These observations identify ODB1 as a further component of homologous recombination-dependent DNA repair in plant mitochondria.

Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

Recruitment of AtWHY1 and AtWHY3 by a distal element upstream of the kinesin gene AtKP1 to mediate transcriptional repression.

A 43-bp distal element, the AtKP1-related element (KPRE), was previously shown to repress the promoter activity of the kinesin gene AtKP1 in Arabidopsis thaliana. In order to identify KPRE-binding factor 1 (KBF1), a combination of ion-exchange chromatography, gel-filtration chromatography and DNA-affinity chromatography was used to purify KBF1 from whole cell extracts of Arabidopsis seedlings. Mass spectrometric identification showed that KBF1 contains two members of the whirly family of transcription factors, AtWHY1 and AtWHY3. KBF1 is a single and double-stranded DNA-binding factor. A ChIP assay showed that AtWHY1 and AtWHY3 bind to the upstream region of AtKP1 gene in vivo. Over-expression of AtWHY1 and AtWHY3 led to an obvious decrease of AtKP1 transcripts, based on quantitative real-time PCR analysis. Interestingly, salicylic acid treatment resulted in an increase of AtWHY1 and AtWHY3 transcripts, and a decrease of AtKP1 transcripts. Thus, AtWHY1 and AtWHY3, as two components of KBF1, can be recruited at the KPRE site to mediate the transcriptional repression of AtKP1. Our results prove that AtKP1 is a new downstream target of the whirly family of transcription factors.

Purification, crystallization and preliminary X-ray diffraction analysis of the Whirly domain of StWhy2 in complex with single-stranded DNA

StWhy1 and StWhy2 are members of the Whirly family of single-stranded DNA (ssDNA) binding proteins. To understand the mode of binding of the Whirly proteins to single-stranded DNA, crystals of the Whirly domains of both StWhy1 and StWhy2 in complex with single-stranded DNA were obtained by the hanging-drop vapour-diffusion method. The diffraction patterns of the StWhy1-ssDNA complex crystals displayed severe anisotropy and were of low resolution, making them unsuitable for structure determination. In contrast, the crystals of the StWhy2-ssDNA complex diffracted isotropically to 2.20 A resolution. The crystallization and data collection to 2.20 A resolution of StWhy2 in the free form are also reported.

A member of the Whirly family is a multifunctional RNA- and DNA-binding protein that is essential for chloroplast biogenesis.

‘Whirly’ proteins comprise a plant-specific protein family whose members have been described as DNA-binding proteins that influence nuclear transcription and telomere maintenance, and that associate with nucleoids in chloroplasts and mitochondria. We identified the maize WHY1 ortholog among proteins that coimmunoprecipitate with CRS1, which promotes the splicing of the chloroplast atpF group II intron. ZmWHY1 localizes to the chloroplast stroma and to the thylakoid membrane, to which it is tethered by DNA. Genome-wide coimmunoprecipitation assays showed that ZmWHY1 in chloroplast extract is associated with DNA from throughout the plastid genome and with a subset of plastid RNAs that includes atpF transcripts. Furthermore, ZmWHY1 binds both RNA and DNA in vitro. A severe ZmWhy1 mutant allele conditions albino seedlings lacking plastid ribosomes; these exhibit the altered plastid RNA profile characteristic of ribosome-less plastids. Hypomorphic ZmWhy1 mutants exhibit reduced atpF intron splicing and a reduced content of plastid ribosomes; aberrant 23S rRNA metabolism in these mutants suggests that a defect in the biogenesis of the large ribosomal subunit underlies the ribosome deficiency. However, these mutants contain near normal levels of chloroplast DNA and RNAs, suggesting that ZmWHY1 is not directly required for either DNA replication or for global plastid transcription.

DNA-binding proteins of the Whirly family in Arabidopsis thaliana are targeted to the organelles

Arabidopsis thaliana contains three genes with high homology to potato p24 which was described as a member of the Whirly family of nuclear transcriptional activators. Computer-based analysis revealed that all Arabidopsis Whirly (Why) proteins contain targeting sequences for either plastids or mitochondria. The functionality of these sequences was demonstrated by in vitro import assays into isolated organelles. Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. The possession of organellar targeting sequences seems to be conserved among Why proteins of higher plant species, including potato p24.

A new family of plant transcription factors displays a novel ssDNA-binding surface.

The crystal structure of p24, the single-stranded DNA (ssDNA) binding subunit of the plant defense transcription factor PBF-2, has been determined to 2.3 A resolution. p24 is representative of a novel family of ubiquitous plant-specific proteins that we refer to as the Whirly family because of their quaternary structure. PBF-2 is composed of four p24 molecules that interact through a helix-loop-helix motif. This interaction produces a central pore, with beta-strands radiating outwards, resulting in a whirligig appearance to the quaternary structure. The noncrystallographic C(4) symmetry arrangement of p24 subunits is novel for ssDNA binding proteins and may explain the binding specificity of PBF-2. This structural arrangement also supports the role of PBF-2 in binding melted promoter regions to modulate gene expression.