Search Results (13)

With the continuous change of climate, drought stress has emerged as the primary constraint on crop growth, posing a significant threat to the stability of global grain reserves. Arbuscular mycorrhizal fungi (AMF), as a kind of widely distributed root endophytes, enhance the drought tolerance of maize (Zea mays L.) through regulating the physiological and molecular responses. However, comprehensive transcriptome analysis to reveal the molecular mechanism of drought tolerance in the symbiotic process between AMF and maize is still limited. In the potted plant experiment, maizes inoculated with and without arbuscular mycorrhizal fungus Funneliformis mosseae were grown under well-watered (WW) or drought-stressed (DS) conditions. By using RNA-Seq and transcriptome analysis on maize roots and leaves, this work aimed to investigate the differential expressed genes (DEGs) related to the Ca²⁺ signaling pathway induced by AMF symbiosis under drought stress. Our findings indicated that F. mosseae inoculation resulted in a decrease in the net fluxes of Ca²⁺, while simultaneously elevating Ca²⁺ contents in the maize roots and leaves under well-watered or drought-stressed conditions. Notably, 189 DEGs were regulated not only by AMF symbiosis and drought stress, but also exhibited preferential expression in either leaves or roots. The annotation and enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that most of the DEGs were significantly enriched in Ca²⁺ signaling pathway genes, related to signal transduction, cellular process, and defense response. A high number of DEGs with this function (including calcineurin B-like protein (CBL), CBL-interacting protein kinase (CIPK), mitogen-activated protein kinase (MAPK), and calcium-dependent protein kinase (CDPK) receptor kinases) were upregulated-DEGs or downregulated-DEGs in F. mosseae-inoculated maizes under drought stress. Furthermore, some DEGs belong to transcription factor (TF) families, including bHLH ERF, and, MYB, were speculated to play key roles in improving the drought tolerance of maize. Based on the expression data and co-expression analysis between TF and Ca²⁺ signaling pathway genes, Whirly1 with CBL11, and BRI1-EMS-SUPPRESSOR 1 (BES1) with CBL10, CIPK24, CDPK1, CDPK14, CDPK19, and MAPK9 genes showed significant positive correlations, while B3 domain-containing transcription factors (B3 TFs) with MAPK1 and both CBL9 genes showed significant negative correlations in response to both F. mosseae inoculation and drought stress. The regulation of Ca²⁺ signaling pathways by AMF symbiosis was an important response mechanism of maize to improve their drought resistance. This study provides insightful perspectives on how AMF-induced modulation of gene expression within the Ca²⁺ signaling pathway can enhance the drought tolerance of mycorrhizal maize in the future. Full article

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. Full article

Sweet cherry (Prunus avium) is one of the economically valuable horticultural fruit trees and it is widely cultivated throughout the world. Whirly (WHY) genes are a unique gene family with few members and have important biological functions in plant growth, development, and response to abiotic stress. This study utilized whole-genome identification to conduct a comprehensive analysis of the WHY genes in sweet cherry and examined their transcription levels in different tissues and under abiotic stress to explore their functions. Two WHY genes were identified in the sweet cherry genome and named PavWHY1 and PavWHY2, respectively, based on their homology with those in Arabidopsis thaliana. Both genes have theoretical isoelectric points greater than seven and are hydrophilic proteins, suggesting that they may be localized in plastids. The two genes are evolutionarily classified into two categories, with large differences in gene structure, and highly similar protein tertiary structures, and both have conserved domains of WHY. PavWHY1 and PavWHY2 are collinear with AtWHY1 and AtWHY2, respectively. The promoter sequence contains cis-acting elements related to hormones and abiotic stress, which are differentially expressed during flower bud differentiation, fruit development, and cold accumulation. qRT–PCR showed that PavWHY1 and PavWHY2 were differentially expressed in flower and fruit development and responded to low temperature and exogenous ABA treatment. The recombinant plasmid pGreenII-0800-Luc with the promoters of these two genes can activate luciferase expression in tobacco. Protein interaction predictions indicate that these gene products may interact with other proteins. This study reveals the molecular features, evolutionary relationships, and expression patterns of sweet cherry WHY genes, and investigates the activities of their promoters, which lays the foundation for further exploration of their biological functions and provides new insights into the WHY gene family in Rosaceae. Full article

The Whirly (WHY) gene family, functioning as transcription factors, plays an essential role in the regulation of plant metabolic responses, which has been demonstrated across multiple species. However, the WHY gene family and its functions in soybean remains unclear. In this paper, we conducted genome-wide screening and identification to characterize the WHY gene family. Seven WHY members were identified and randomly distributed across six chromosomes. The phylogenetic evolutionary tree of WHY genes in soybean and other species was divided into five clades. An in-depth analysis revealed that segmental duplications significantly contributed to the expansion of GmWHYs, and the GmWHY gene members may have experienced evolutionary pressure for purifying selection in soybeans. The analysis of promoter Cis-elements in GmWHYs suggested their potential significance in addressing diverse stress conditions. The expression patterns of GmWHYs exhibited tissue-specific variations throughout the different stages of soybean development. Additionally, six GmWHY genes exhibited different responses to low phosphate stress. These findings will provide a theoretical basis and valuable reference for the future exploration of WHY gene function. Full article

WHIRLY1, a small plant-specific ssDNA-binding protein, dually located in chloroplasts and the nucleus, is discussed to act as a retrograde signal transmitting a stress signal from the chloroplast to the nucleus and triggering there a stress-related gene expression. In this work, we investigated the function of WHIRLY1 in the drought stress response of barley, employing two overexpression lines (oeW1-2 and oeW1-15). The overexpression of WHIRLY1 delayed the drought-stress-related onset of senescence in primary leaves. Two abscisic acid (ABA)-dependent marker genes of drought stress, HvNCED1 and HvS40, whose expression in the wild type was induced during drought treatment, were not induced in overexpression lines. In addition, a drought-related increase in ABA concentration in the leaves was suppressed in WHIRLY1 overexpression lines. To analyze the impact of the gain-of-function of WHIRLY1 on the drought-related reprogramming of nuclear gene expression, RNAseq was performed comparing the wild type and an overexpression line. Cluster analyses revealed a set of genes highly up-regulated in response to drought in the wild type but not in the WHIRLY1 overexpression lines. Among these genes were many stress- and abscisic acid (ABA)-related ones. Another cluster comprised genes up-regulated in the oeW1 lines compared to the wild type. These were related to primary metabolism, chloroplast function and growth. Our results indicate that WHIRLY1 acts as a hub, balancing trade-off between stress-related and developmental pathways. To test whether the gain-of-function of WHIRLY1 affects the epigenetic control of stress-related gene expression, we analyzed drought-related histone modifications in different regions of the promoter and at the transcriptional start sites of HvNCED1 and HvS40. Interestingly, the level of euchromatic marks (H3K4me3 and H3K9ac) was clearly decreased in both genes in a WHIRLY1 overexpression line. Our results indicate that WHIRLY1, which is discussed to act as a retrograde signal, affects the ABA-related reprogramming of nuclear gene expression during drought via differential histone modifications. Full article

Vitis vinifera plants are disease-susceptible while Vitis pseudoreticulata plants are disease-resistant; however, the molecular mechanism remains unclear. In this study, the single-stranded DNA- and RNA-binding protein gene Whirly (VvWhy1 and VpWhy1) were cloned from V. vinifera “Cabernet Sauvignon” and V. pseudoreticulata “HD1”. VvWhy1 and VpWhy1 promoter sequences (pVv and pVp) were also isolated; however, the identity of the promoter sequences was far lower than that between the Why1 coding sequences (CDSs). Both Why1 gene sequences had seven exons and six introns, and they had a C-terminal Whirly conserved domain and N-terminal chloroplast transit peptide, which was then verified to be chloroplast localization. Transcriptional expression showed that VpWhy1 was strongly induced by Plasmopara viticola, while VvWhy1 showed a low expression level. Further, the GUS activity indicated pVp had high activity involved in response to Phytophthora capsici infection. In addition, Nicotiana benthamiana transiently expressing pVp::VvWhy1 and pVp::VpWhy1 enhanced the P. capsici resistance. Moreover, Why1, PR1 and PR10 were upregulated in pVp transgenic N. benthamiana leaves. This research presented a novel insight into disease resistance mechanism that pVp promoted the transcription of Why1, which subsequently regulated the expression of PR1 and PR10, further enhancing the resistance to P. capsici. Full article

Among abiotic stressors, drought and salinity seriously affect crop growth worldwide. In plants, research has aimed to increase stress-responsive protein synthesis upstream or downstream of the various transcription factors (TFs) that alleviate drought and salinity stress. TFs play diverse roles in controlling gene expression in plants, which is necessary to regulate biological processes, such as development and environmental stress responses. In general, plant responses to different stress conditions may be either abscisic acid (ABA)-dependent or ABA-independent. A detailed understanding of how TF pathways and ABA interact to cause stress responses is essential to improve tolerance to drought and salinity stress. Despite previous progress, more active approaches based on TFs are the current focus. Therefore, the present review emphasizes the recent advancements in complex cascades of gene expression during drought and salinity responses, especially identifying the specificity and crosstalk in ABA-dependent and -independent signaling pathways. This review also highlights the transcriptional regulation of gene expression governed by various key TF pathways, including AP2/ERF, bHLH, bZIP, DREB, GATA, HD-Zip, Homeo-box, MADS-box, MYB, NAC, Tri-helix, WHIRLY, WOX, WRKY, YABBY, and zinc finger, operating in ABA-dependent and -independent signaling pathways. Full article

Plastid-nucleus retrograde signaling (PNRS) play essential roles in regulating nuclear gene expression during plant growth and development. Excessive reactive oxygen species can trigger PNRS. We previously reported that in apple (Malus domestica Borkh.) seedlings, the expression of microRNA156 (miR156) was significantly low in the adult phase, which was accompanied by high levels of hydrogen peroxide (H₂O₂) accumulation in chloroplasts. However, it was unclear whether adult-phase-specific chloroplast H₂O₂ may induce PNRS and affect miR156 expression, or miR156 triggers adult phase PNRS during the ontogenesis. In this paper, we examined the relationship between miR156 levels and six PNRS components in juvenile and adult phase leaves from ‘Zisai Pearl’בRed Fuji’ hybrids. We found that PNRS generated by singlet oxygen (¹O₂), the photosynthetic redox state, methylerythritol cyclodiphosphate (MEcPP), SAL1-3-phosphoadenosine 5-phosphate (PAP) and WHIRLY1 were not involved. The accumulation of Mg-protoporphyrin IX (Mg-Proto IX), the expression of the synthetic genes MdGUN5 and MdGUN6, and Mg-Proto IX PNRS related nuclear genes increased with ontogenesis. These changes were negatively correlated with miR156 expression. Manipulating Mg-Proto IX synthesis with 5-aminolevulinic acid (ALA) or gabaculine did not affect miR156 expression in vitro shoots. In contrast, modulating miR156 expression via MdGGT1 or MdMIR156a6 transgenesis led to changes in Mg-Proto IX contents and the corresponding gene expressions. It was concluded that the Mg-Proto IX PNRS was regulated downstream of miR156 regardless of adult-phase-specific plastid H₂O₂ accumulation. The findings may facilitate the understanding of the mechanism of ontogenesis in higher plants. Full article

Leaf senescence, either as a natural stage of development or as an induced process under stress conditions, incorporates multiple intricate signaling pathways. At the cellular level, retrograde signals have been considered as important players during the initiation and progression of senescence in both animals and plants. The plant-specific single-strand DNA-binding protein WHIRLY1 (WHY1), a repressor of leaf natural senescence, is dually located in both nucleus and plastids. Despite many years of studies, the myth about its dual location and the underlying functional implications remain elusive. Here, we provide further evidence in Arabidopsis showing that alteration in WHY1 allocation between the nucleus and chloroplast causes perturbation in H₂O₂ homeostasis, resulting in adverse plant senescence phenotypes. The knockout of WHY1 increased H₂O₂ content at 37 days post-germination, coincident with an early leaf senescence phenotype, which can be rescued by ectopic expression of the nuclear isoform (nWHY1), but not by the plastid isoform (pWHY1). Instead, accumulated pWHY1 greatly provoked H₂O₂ in cells. On the other hand, exogenous H₂O₂ treatment induced a substantial plastid accumulation of WHY1 proteins and at the same time reduced the nuclear isoforms. This H₂O₂-induced loss of nucleus WHY1 isoform was accompanied by enhanced enrichments of histone H3 lysine 9 acetylation (H3K9ac) and recruitment of RNA polymerase II (RNAP II) globally, and specifically at the promoter of the senescence-related transcription factor WRKY53, which in turn activated WRKY53 transcription and led to a senescence phenotype. Thus, the distribution of WHY1 organelle isoforms and the feedback of H₂O₂ intervene in a circularly integrated regulatory network during plant senescence in Arabidopsis. Full article

Pale yellowing of leaf variegation is observed in the mutant Arabidopsis lines Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) overexpression (oeCIPK14) and double-knockout WHIRLY1/WHIRLY3 (why1/3). Further, the relative distribution of WHIRLY1 (WHY1) protein between plastids and the nucleus is affected by the phosphorylation of WHY1 by CIPK14. To elucidate the coregulation of CIPK14 and WHIRLY1/WHIRLY3-mediated pale yellowing of leaves, a differential proteomic analysis was conducted between the oeCIPK14 variegated (oeCIPK14-var) line, why1/3 variegated (why1/3-var) line, and wild type (WT). More than 800 protein spots were resolved on each gel, and 67 differentially abundant proteins (DAPs) were identified by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS). Of these 67 proteins, 34 DAPs were in the oeCIPK14-var line and 33 DAPs were in the why1/3-var line compared to the WT. Five overlapping proteins were differentially expressed in both the oeCIPK14-var and why1/3-var lines: ATP-dependent Clp protease proteolytic subunit-related protein 3 (ClpR3), Ribulose bisphosphate carboxylase large chain (RBCL), Beta-amylase 3 (BAM3), Ribosome-recycling factor (RRF), and Ribulose bisphosphate carboxylase small chain (RBCS). Bioinformatics analysis showed that most of the DAPs are involved in photosynthesis, defense and antioxidation pathways, protein metabolism, amino acid metabolism, energy metabolism, malate biosynthesis, lipid metabolism, and transcription. Thus, in the why1/3-var and oeCIPK14-var lines, there was a decrease in the photosystem parameters, including the content of chlorophyll, the photochemical efficiency of photosystem (PS II) (F_v/F_m), and electron transport rates (ETRs), but there was an increase in non-photochemical quenching (NPQ). Both mutants showed high sensitivity to intense light. Based on the annotation of the DAPs from both why1/3-var and oeCIPK14-var lines, we conclude that the CIPK14 phosphorylation-mediated WHY1 deficiency in plastids is related to the impairment of protein metabolism, leading to chloroplast dysfunction. Full article

Plastid-nucleus-located WHIRLY1 protein plays a role in regulating leaf senescence and is believed to associate with the increase of reactive oxygen species delivered from redox state of the photosynthetic electron transport chain. In order to make sure whether WHIRLY1 plays a role in photosynthesis, in this study, the performances of photosynthesis were detected in Arabidopsis whirly1 knockout (kowhy1) and plastid localized WHIRLY1 overexpression (oepWHY1) plants. Loss of WHIRLY1 leads to a higher photochemical quantum yield of photosystem I Y(I) and electron transport rate (ETR) and a lower non-photochemical quenching (NPQ) involved in the thermal dissipation of excitation energy of chlorophyll fluorescence than the wild type. Further analyses showed that WHIRLY1 interacts with Light-harvesting protein complex I (LHCA1) and affects the expression of genes encoding photosystem I (PSI) and light harvest complexes (LHCI). Moreover, loss of WHIRLY1 decreases chloroplast NAD(P)H dehydrogenase-like complex (NDH) activity and the accumulation of NDH supercomplex. Several genes encoding the PSI-NDH complexes are also up-regulated in kowhy1 and the whirly1whirly3 double mutant (ko1/3) but steady in oepWHY1 plants. However, under high light conditions (800 μmol m⁻² s⁻¹), both kowhy1 and ko1/3 plants show lower ETR than wild-type which are contrary to that under normal light condition. Moreover, the expression of several PSI-NDH encoding genes and ERF109 which is related to jasmonate (JA) response varied in kowhy1 under different light conditions. These results indicate that WHIRLY1 is involved in the alteration of ETR by affecting the activities of PSI and supercomplex formation of PSI with LHCI or NDH and may acting as a communicator between the plastids and the nucleus. Full article

The plastid-nucleus located protein WHIRLY1 has been described as an upstream regulator of leaf senescence, binding to the promoter of senescence-associated genes like HvS40. To investigate the impact of WHIRLY1 on drought stress-induced, premature senescence, transgenic barley plants with an RNAi-mediated knockdown of the HvWHIRLY1 gene were grown under normal and drought stress conditions. The course of leaf senescence in these lines was monitored by physiological parameters and studies on the expression of senescence- and drought stress-related genes. Drought treatment accelerated leaf senescence in WT plants, whereas WHIRLY 1 knockdown lines (RNAi-W1) showed a stay-green phenotype. Expression of both senescence-associated and drought stress-responsive genes, was delayed in the transgenic plants. Notably, expression of transcription factors of the WRKY and NAC families, which are known to function in senescence- and stress-related signaling pathways, was affected in plants with impaired accumulation of WHIRLY1, indicating that WHIRLY1 acts as an upstream regulator of drought stress-induced senescence. To reveal the epigenetic indexing of HvS40 at the onset of drought-induced senescence in WT and RNAi-W1 lines, stress-responsive loading with histone modifications of promoter and coding sequences of HvS40 was analyzed by chromatin immunoprecipitation and quantified by qRT-PCR. In the wildtype, the euchromatic mark H3K9ac of the HvS40 gene was low under control conditions and was established in response to drought treatment, indicating the action of epigenetic mechanisms in response to drought stress. However, drought stress caused no significant increase in H3K9ac in plants impaired in accumulation of WHIRLY1. The results show that WHIRLY1 knockdown sets in motion a delay in senescence that involves all aspects of gene expression, including changes in chromatin structure. Full article

Changes in the developmental or metabolic state of plastids can trigger profound changes in the transcript profiles of nuclear genes. Many nuclear transcription factors were shown to be controlled by signals generated in the organelles. In addition to the many different compounds for which an involvement in retrograde signaling is discussed, accumulating evidence suggests a role for proteins in plastid-to-nucleus communication. These proteins might be sequestered in the plastids before they act as transcriptional regulators in the nucleus. Indeed, several proteins exhibiting a dual localization in the plastids and the nucleus are promising candidates for such a direct signal transduction involving regulatory protein storage in the plastids. Among such proteins, the nuclear transcription factor WHIRLY1 stands out as being the only protein for which an export from plastids and translocation to the nucleus has been experimentally demonstrated. Other proteins, however, strongly support the notion that this pathway might be more common than currently believed. Full article