CUP-SHAPED COTYLEDON Research Articles

Transcription Factor RhCUC3 Regulates Petal Numbers in Rose Flowers

Rose is one of the most popular ornamental plants worldwide. The double-flower trait, referring to flowers with extra petals, has been a key focus in rose breeding history. However, the genetic mechanisms regulating petal number in roses are still not fully understood. Here, we identified the CUP-SHAPED COTYLEDON 3 (RhCUC3) gene in the miniature rose (Rosa hybrida ‘Eclair’). The expression of RhCUC3 was high during the petal and stamen primordium differentiation stages but declined sharply during pistil primordium development. RhCUC3 belongs to the NAM/CUC3 subgroup of NAC transcription factors and is localized in the nucleus. The transcript level of RhCUC3 increased significantly with ABA and GA treatments and was inversely down-regulated with MeJA and 6-BA treatments. Silencing RhCUC3 using virus-induced gene silencing (VIGS) in rose ‘Eclair’ significantly decreased the number of petaloid stamens and normal petals while slightly increasing the number of stamens. Additionally, the expression of RhAG and RhAGL, two MADS-box genes associated with floral organ identity, was significantly higher in TRV-RhCUC3 compared to the TRV control. These findings suggest that RhCUC3 enhances stamen petaloidy and petal number, potentially by modulating the expression of RhAG and RhAGL, providing new insights into the function of NAC transcription factors in plants.

Inhibition of histone deacetylase in Arabidopsis root calli promotes de novo shoot organogenesis

De novo organogenesis from somatic cells to the entire plant represents a remarkable biological phenomenon, but the underlying regulatory mechanism, particularly at the epigenetic level, remains obscure. In this work, we demonstrate the important role of histone deacetylases (HDACs) in shoot organogenesis. HDAC inhibition by trichostatin A (an HDAC inhibitor) at the callus induction stage promotes shoot formation in wounded roots and circumvents tissue wounding to initiate shoot regeneration in unwounded roots. This HDAC inhibition-mediated promotion of shoot organogenesis in wounded roots is associated with the concomitant upregulation of the wound signaling pathway (WOUND INDUCED DEDIFFERENTIATION 4, ENHANCER OF SHOOT REGENERATION1, ISOPENTENYLTRANSFERASE 5, CUP-SHAPED COTYLEDON 2 etc.) and the ARF-LBD pathway (AUXIN RESPONSE FACTOR 19, LATERAL ORGAN BOUNDARIES-DOMAIN 29, etc.) and the downregulation of auxin biosynthesis and reduced auxin content. Furthermore, inhibiting HDACs enhances the local enrichment of histone 3 lysine 9/lysine 14 acetylation at ISOPENTENYLTRANSFERASE 5, supporting the role of histone acetylation in its transcriptional regulation. On the other hand, the HDAC inhibition-associated activation of shoot organogenesis from unwounded roots is coupled with increased expression of the ARF-LBD pathway gene LATERAL ORGAN BOUNDARIES-DOMAIN 29 while bypassing the wound signaling or auxin biosynthetic genes. These findings provide novel insights into the regulatory mechanisms underlying de novo shoot organogenesis and lay a foundation for the improvement of plant transformation technologies.

Deciphering Drought Resilience in Solanaceae Crops: Unraveling Molecular and Genetic Mechanisms.

The Solanaceae family, which includes vital crops such as tomatoes, peppers, eggplants, and potatoes, is increasingly impacted by drought due to climate change. Recent research has concentrated on unraveling the molecular mechanisms behind drought resistance in these crops, with a focus on abscisic acid (ABA) signaling pathways, transcription factors (TFs) like MYB (Myeloblastosis), WRKY (WRKY DNA-binding protein), and NAC (NAM, ATAF1/2, and CUC2- NAM: No Apical Meristem, ATAF1/2, and CUC2: Cup-shaped Cotyledon), and the omics approaches. Moreover, transcriptome sequencing (RNA-seq) has been instrumental in identifying differentially expressed genes (DEGs) crucial for drought adaptation. Proteomics studies further reveal changes in protein expression under drought conditions, elucidating stress response mechanisms. Additionally, microRNAs (miRNAs) have been identified as key regulators in drought response. Advances in proteomics and transcriptomics have highlighted key proteins and genes that respond to drought stress, offering new insights into drought tolerance. To address the challenge of drought, future research should emphasize the development of drought-resistant varieties through precision breeding techniques such as gene editing, marker-assisted selection (MAS), and the integration of artificial intelligence. Additionally, the adoption of environmentally sustainable cultivation practices, including precision irrigation and the use of anti-drought agents, is crucial for improving water-use efficiency and crop resilience. International collaboration and data sharing will be essential to accelerate progress and ensure global food security in increasingly arid conditions. These efforts will enable Solanaceae crops to adapt the challenges posed by climate change, ensuring their productivity and sustainability.

DET1 modulates ATAF1-repressed thermosensory elongation through ubiquitination in Arabidopsis.

The Arabidopsis transcription factor ATAF1 negatively regulates thermomorphogenesis by inhibiting the expression of key genes involved in thermoresponsive elongation. DET1-mediated ubiquitination promotes ATAF1 degradation. In response to warmer, non-stressful average temperatures, plants have evolved an adaptive morphologic response called thermomorphogenesis to increase their fitness. This adaptive morphologic development is regulated by transcription factors (TFs) that control the expression of heat-induced genes that gate thermoresponsive growth. No apical meristem (NAM), Arabidopsis thaliana-activating factor 1/2 (ATAF1/2), and cup-shaped cotyledon 2 (CUC2) (collectively known as NAC) TFs regulate morphogenesis and respond to temperature stress, but whether they regulate thermomorphogenesis remains largely unknown. Here, we identified ATAF1 as a negative regulator of thermomorphogenesis and revealed that the E3-ligase component de-etiolated 1 (DET1) mediated ATAF1 ubiquitination and degradation. Our results revealed that ATAF1 negatively regulates warm temperature-induced hypocotyl elongation and inhibits the expression of thermoresponsive genes. Moreover, ATAF1 directly targeted and repressed the expression of YUCCA 8 (YUC8) and phytochrome interacting factor 4 (PIF4), two key regulators involved in elongation. At the post-translational level, elevated ambient temperatures negatively modulated the stability of ATAF1 by inducing the DET1-mediated ubiquitination pathway. Our results demonstrated the presence of a DET1-ATAF1-PIF4/YUC8 control module for thermomorphogenesis in plants, which may increase fitness by fine-tuning thermoresponsive gene expression under warm temperatures.

Evolution of NAC transcription factors from early land plants to domesticated crops

Abstract NAC [NO APICAL MERISTEM (NAM), ARABIDOPSIS TRANSCRIPTION ACTIVATOR FACTOR 1/2 (ATAF1/2), and CUP-SHAPED COTYLEDON (CUC2)] transcription factors are key regulators of plant growth, development, and stress responses but were also crucial players during land plant adaptation and crop domestication. Using representative members of green algae, bryophytes, lycophytes, gymnosperms, and angiosperms, we expanded the evolutionary history of NAC transcription factors to unveil the relationships among members of this gene family. We found a massive increase in the number of NAC transcription factors from green algae to lycophytes and an even larger increase in flowering plants. Many of the NAC clades arose later during evolution since we found eudicot- and monocot-specific clades. Cis-elements analysis in NAC promoters showed the presence of abiotic and biotic stress as well as hormonal response elements, which indicate the ancestral function of NAC transcription factor genes in response to environmental stimuli and in plant development. At the transcriptional level, the expression of NAC transcription factors was low or absent in male reproduction, particularly mature pollen, across the plant kingdom. We also identified NAC genes with conserved expression patterns in response to heat stress in Marchantia polymorpha and Oryza sativa. Our study provides further evidence that transcriptional mechanisms associated with stress responses and development emerged early during plant land adaptation and are still conserved in flowering plants and domesticated crops.

WOX1 controls leaf serration development via temporally restricting BRASSINAZOLE RESISTANT 1 and CUP SHAPED COTYLEDON 3 expression in Arabidopsis.

Leaves have evolved shape diversity, ranging from simple leaves with a smooth margin to complicated shapes with toothed/serrated, lobed, and dissected leaves with leaflets. In the model plant Arabidopsis thaliana with simple leaves producing a serrated margin, boundary regulatory factor genes CUP SHAPED COTYLEDON 2 (CUC2) and CUC3 play important roles in promoting leaf initiation and maintenance of serration. Stem cell-related WUSCHEL-RELATED HOMEOBOX1 (WOX1) and PRESSED FLOWER (PRS)/WOX3 are also essential for leaf margin morphogenesis, but the role of WOX1 and PRS as well as the relationships between CUCs and WOXs for tooth development are unclear. In this study, we found that WOX1, but not PRS, prevents overproduction of number of teeth and excessive tooth size by limiting CUC3 expression to a moderate level in a temporally regulated manner. We also revealed that BRASSINAZOLE RESISTANT 1 (BZR1), a known regulator of plant development including boundary regions, is involved in WOX1 negative regulation of tooth development by repressing CUC3 expression during the initiation/early stage of tooth development. WOX1 parallelly limits BZR1 and CUC3 expression from the late stage of the first two teeth, while it restricts CUC3 activity in a BZR1-dependent manner from the initiation/early stage of subsequently developed teeth. This study uncovers a new mechanism for WOX1 in fine-tuning the leaf margin geometry.

MIR164B ensures robust Arabidopsis leaf development by compensating for compromised POLYCOMB REPRESSIVE COMPLEX2 function.

Robustness is pervasive throughout biological systems, enabling them to maintain persistent outputs despite perturbations in their components. Here, we reveal a mechanism contributing to leaf morphology robustness in the face of genetic perturbations. In Arabidopsis (Arabidopsis thaliana), leaf shape is established during early development through the quantitative action of the CUP-SHAPED COTYLEDON2 (CUC2) protein, whose encoding gene is negatively regulated by the co-expressed MICRORNA164A (MIR164A) gene. Compromised epigenetic regulation due to defective Polycomb Repressive Complex 2 (PRC2) function results in the transcriptional derepression of CUC2 but has no impact on CUC2 protein dynamics or early morphogenesis. We solve this apparent paradox by showing that compromised PRC2 function simultaneously derepresses the expression of another member of the MIR164 gene family, MIR164B. This mechanism dampens CUC2 protein levels, thereby compensating for compromised PRC2 function and canalizing early leaf morphogenesis. Furthermore, we show that this compensation mechanism is active under different environmental conditions. Our findings shed light on how the interplay between different steps of gene expression regulation can contribute to developmental robustness.

GhSBI1, a CUP-SHAPED COTYLEDON 2 homologue, modulates branch internode elongation in cotton.

Branch length is an important plant architecture trait in cotton (Gossypium) breeding. Development of cultivars with short branch has been proposed as a main object to enhance cotton yield potential, because they are suitable for high planting density. Here, we report the molecular cloning and characterization of a semi-dominant quantitative trait locus, Short Branch Internode 1(GhSBI1), which encodes a NAC transcription factor homologous to CUP-SHAPED COTYLEDON 2 (CUC2) and is regulated by microRNA ghr-miR164. We demonstrate that a point mutation found in sbi1 mutants perturbs ghr-miR164-directed regulation of GhSBI1, resulting in an increased expression level of GhSBI1. The sbi1 mutant was sensitive to exogenous gibberellic acid (GA) treatments. Overexpression of GhSBI1 inhibited branch internode elongation and led to the decreased levels of bioactive GAs. In addition, gene knockout analysis showed that GhSBI1 is required for the maintenance of the boundaries of multiple tissues in cotton. Transcriptome analysis revealed that overexpression of GhSBI1 affects the expression of plant hormone signalling-, axillary meristems initiation-, and abiotic stress response-related genes. GhSBI1 interacted with GAIs, the DELLA repressors of GA signalling. GhSBI1 represses expression of GA signalling- and cell elongation-related genes by directly targeting their promoters. Our work thus provides new insights into the molecular mechanisms for branch length and paves the way for the development of elite cultivars with suitable plant architecture in cotton.

Tradeoff between speed and robustness in primordium initiation mediated by auxin-CUC1 interaction

Robustness is the reproducible development of a phenotype despite stochastic noise. It often involves tradeoffs with other performance metrics, but the mechanisms underlying such tradeoffs were largely unknown. An Arabidopsis flower robustly develops four sepals from four precisely positioned auxin maxima. The development related myb-like 1 (drmy1) mutant generates noise in auxin signaling that disrupts robustness in sepal initiation. Here, we find that increased expression of CUP-SHAPED COTYLEDON1 (CUC1), a boundary specification transcription factor, in drmy1 underlies this loss of robustness. CUC1 surrounds and amplifies stochastic auxin noise in drmy1 to form variably positioned auxin maxima and sepal primordia. Removing CUC1 from drmy1 provides time for noisy auxin signaling to resolve into four precisely positioned auxin maxima, restoring robust sepal initiation. However, removing CUC1 decreases the intensity of auxin maxima and slows down sepal initiation. Thus, CUC1 increases morphogenesis speed but impairs robustness against auxin noise. Further, using a computational model, we find that the observed phenotype can be explained by the effect of CUC1 in repolarizing PIN FORMED1 (PIN1), a polar auxin transporter. Lastly, our model predicts that reducing global growth rate improves developmental robustness, which we validate experimentally. Thus, our study illustrates a tradeoff between speed and robustness during development.

A CUC1/auxin genetic module links cell polarity to patterned tissue growth and leaf shape diversity in crucifer plants

How tissue-level information encoded by fields of regulatory gene activity is translated into the patterns of cell polarity and growth that generate the diverse shapes of different species remains poorly understood. Here, we investigate this problem in the case of leaf shape differences between Arabidopsis thaliana, which has simple leaves, and its relative Cardamine hirsuta that has complex leaves divided into leaflets. We show that patterned expression of the transcription factor CUP-SHAPED COTYLEDON1 in C. hirsuta (ChCUC1) is a key determinant of leaf shape differences between the two species. Through inducible genetic perturbations, time-lapse imaging of growth, and computational modeling, we find that ChCUC1 provides instructive input into auxin-based leaf margin patterning. This input arises via transcriptional regulation of multiple auxin homeostasis components, including direct activation of WAG kinases that are known to regulate the polarity of PIN-FORMED auxin transporters. Thus, we have uncovered a mechanism that bridges biological scales by linking spatially distributed and species-specific transcription factor expression to cell-level polarity and growth, to shape diverse leaf forms.

Tradeoff Between Speed and Robustness in Primordium Initiation Mediated by Auxin-CUC1 Interaction.

Robustness is the reproducible development of a phenotype despite stochastic noise. It often involves tradeoffs with other performance metrics, but the mechanisms underlying such tradeoffs were largely unknown. An Arabidopsis flower robustly develops four sepals from four precisely positioned auxin maxima. The development related myb-like 1 (drmy1) mutant generates noise in auxin signaling that disrupts robustness in sepal initiation. Here, we found that increased expression of CUP-SHAPED COTYLEDON1 (CUC1), a boundary specification transcription factor, in drmy1 underlies this loss of robustness. CUC1 surrounds and amplifies stochastic auxin noise in drmy1 to form variably positioned auxin maxima and sepal primordia. Removing CUC1 from drmy1 provides time for noisy auxin signaling to resolve into four precisely positioned auxin maxima, restoring robust sepal initiation. However, removing CUC1 decreases auxin maxima intensity and slows down sepal initiation. Thus, CUC1 increases morphogenesis speed but impairs robustness against auxin noise. Further, using a computational model, we found that the observed phenotype can be explained by the effect of CUC1 in repolarizing PIN FORMED1 (PIN1), a polar auxin transporter. Lastly, our model predicts that reducing global growth rate improves developmental robustness, which we validated experimentally. Thus, our study illustrates a tradeoff between speed and robustness during development.

Common Bean (Phaseolus vulgaris L.) NAC Transcriptional Factor PvNAC52 Enhances Transgenic Arabidopsis Resistance to Salt, Alkali, Osmotic, and ABA Stress by Upregulating Stress-Responsive Genes.

The NAC family of transcription factors includes no apical meristem (NAM), Arabidopsis thaliana transcription activator 1/2 (ATAF1/2), and cup-shaped cotyledon (CUC2) proteins, which are unique to plants, contributing significantly to their adaptation to environmental challenges. In the present study, we observed that the PvNAC52 protein is predominantly expressed in the cell membrane, cytoplasm, and nucleus. Overexpression of PvNAC52 in Arabidopsis strengthened plant resilience to salt, alkali, osmotic, and ABA stresses. PvNAC52 significantly (p < 0.05) reduced the degree of oxidative damage to cell membranes, proline content, and plant water loss by increasing the expression of MSD1, FSD1, CSD1, POD, PRX69, CAT, and P5CS2. Moreover, the expression of genes associated with abiotic stress responses, such as SOS1, P5S1, RD29A, NCED3, ABIs, LEAs, and DREBs, was enhanced by PvNAC52 overexpression. A yeast one-hybrid assay showed that PvNAC52 specifically binds to the cis-acting elements ABRE (abscisic acid-responsive elements, ACGTG) within the promoter. This further suggests that PvNAC52 is responsible for the transcriptional modulation of abiotic stress response genes by identifying the core sequence, ACGTG. These findings provide a theoretical foundation for the further analysis of the targeted cis-acting elements and genes downstream of PvNAC52 in the common bean.

A NAC transcription factor represses a module associated with xyloglucan content and regulates aluminum tolerance.

The transcriptional regulation of aluminum (Al) tolerance in plants is largely unknown, although Al toxicity restricts agricultural yields in acidic soils. Here, we identified a NAM, ATAF1/2, and cup-shaped cotyledon 2 (NAC) transcription factor that participates in Al tolerance in Arabidopsis (Arabidopsis thaliana). Al substantially induced the transcript and protein levels of ANAC070, and loss-of-function mutants showed remarkably increased Al sensitivity, implying a beneficial role of ANAC070 in plant tolerance to Al toxicity. Further investigation revealed that more Al accumulated in the roots of anac070 mutants, especially in root cell walls, accompanied by a higher hemicellulose and xyloglucan level, implying a possible interaction between ANAC070 and genes that encode proteins responsible for the modification of xyloglucan, including xyloglucan endo-transglycosylase/hydrolase (XTH) or ANAC017. Yeast 1-hybrid analysis revealed a potential interaction between ANAC070 and ANAC017, but not for other XTHs. Furthermore, dual-luciferase reporter assay, RT-qPCR, and GUS analysis revealed that ANAC070 could directly repress the transcript levels of ANAC017, and knockout of ANAC017 in the anac070 mutant partially restored its Al sensitivity phenotype, indicating that ANAC070 contributes to Al tolerance mechanisms other than suppression of ANAC017 expression. Further analysis revealed that the core transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) and its target genes, which control Al tolerance in Arabidopsis, may also be involved in ANAC070-regulated Al tolerance. In summary, we identified a transcription factor, ANAC070, that represses the ANAC017-XTH31 module to regulate Al tolerance in Arabidopsis.

Expression analyses of CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS in the one-leaf plant Monophyllaea glabra reveal neoteny evolution of shoot meristem

The one-leaf plant Monophyllaea glabra exhibits a unique developmental manner in which only one cotyledon continues growing without producing new vegetative organs. This morphology is formed by specific meristems, the groove meristem (GM) and the basal meristem (BM), which are thought to be modified shoot apical meristem (SAM) and leaf meristem. In this study, we analysed the expression of the organ boundary gene CUP-SHAPED COTYLEDON (CUC) and the SAM maintenance gene SHOOT MERISTEMLESS (STM) orthologs by whole-mount in situ hybridisation. We found that CUCs did not show clear border patterns around GM and BM during the vegetative phase. Furthermore, double-colour detection analysis at the cellular level revealed that CUC and STM expression overlapped in the GM region during the vegetative phase. We also found that this overlap is dissolved in the reproductive phase when normal shoot organogenesis is observed. Since co-expression of these genes occurs during SAM initiation under embryogenesis in Arabidopsis, our results demonstrate that GM is a prolonged stage of pre-mature SAM. Therefore, we propose that neotenic meristems could be a novel plant trait acquired by one-leaf plants.

NAC transcription factor ATAF1 negatively modulates the PIF-regulated hypocotyl elongation under a short-day photoperiod.

Day length modulates hypocotyl elongation in seedlings to optimize their overall fitness. Variations in cell growth-associated genes are regulated by several transcription factors. However, the specific transcription factors through which the plant clock increases plant fitness are still being elucidated. In this study, we identified the no apical meristem, Arabidopsis thaliana-activating factor (ATAF-1/2), and cup-shaped cotyledon (NAC) family transcription factor ATAF1 as a novel repressor of hypocotyl elongation under a short-day (SD) photoperiod. Variations in day length profoundly affected the transcriptional and protein levels of ATAF1. ATAF1-deficient mutant exhibited increased hypocotyl length and cell growth-promoting gene expression under SD conditions. Moreover, ATAF1 directly targeted and repressed the expression of the cycling Dof factor 1/5 (CDF1/5), two key transcription factors involved in hypocotyl elongation under SD conditions. Additionally, ATAF1 interacted with and negatively modulated the effects of phytochrome-interacting factor (PIF), thus inhibiting PIF-promoted gene expression and hypocotyl elongation. Taken together, our results revealed ATAF1-PIF as a crucial pair modulating the expression of key transcription factors to facilitate plant growth during day/night cycles under fluctuating light conditions.

SOG1 and BRCA1 Interdependently Regulate RAD54 Expression for Repairing Salinity-Induced DNA Double-Strand Breaks in Arabidopsis.

As sessile organisms, land plants experience various forms of environmental stresses throughout their life span. Therefore, plants have developed extensive and complicated defense mechanisms, including a robust DNA damage response (DDR) and DNA repair systems for maintaining genome integrity. In Arabidopsis, the NAC [NO APICAL MERISTEM (NAM), ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR (ATAF), CUP-SHAPED COTYLEDON (CUC)] domain family transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) plays an important role in regulating DDR. Here, we show that SOG1 plays a key role in regulating the repair of salinity-induced DNA double-strand breaks (DSBs) via the homologous recombination (HR) pathway in Arabidopsis. The sog1-1 mutant seedlings display a considerably slower rate of repair of salinity-induced DSBs. Accumulation of SOG1 protein increases in wild-type Arabidopsis under salinity stress, and it enhances the expression of HR pathway-related genes, including RAD51, RAD54 and BReast CAncer gene 1 (BRCA1), respectively, as found in SOG1 overexpression lines. SOG1 binds specifically to the AtRAD54 promoter at the 5'-(N)4GTCAA(N)3C-3' consensus sequence and positively regulates its expression under salinity stress. The phenotypic responses of sog1-1/atrad54 double mutants suggest that SOG1 functions upstream of RAD54, and both these genes are essential in regulating DDR under salinity stress. Furthermore, SOG1 interacts directly with BRCA1, an important component of the HR-mediated DSB repair pathway in plants, where BRCA1 appears to facilitate the binding of SOG1 to the RAD54 promoter. At the genetic level, SOG1 and BRCA1 function interdependently in modulating RAD54 expression under salinity-induced DNA damage. Together, our results suggest that SOG1 regulates the repair of salinity-induced DSBs via the HR-mediated pathway through genetic interactions with RAD54 and BRCA1 in Arabidopsis.

A developmentally controlled cellular decompartmentalization process executes programmed cell death in the Arabidopsis root cap.

Programmed cell death (PCD) is a fundamental cellular process crucial to development, homeostasis, and immunity in multicellular eukaryotes. In contrast to our knowledge on the regulation of diverse animal cell death subroutines, information on execution of PCD in plants remains fragmentary. Here, we make use of the accessibility of the Arabidopsis (Arabidopsis thaliana) root cap to visualize the execution process of developmentally controlled PCD. We identify a succession of selective decompartmentalization events and ion fluxes as part of the terminal differentiation program that is orchestrated by the NO APICAL MERISTEM, ARABIDOPSIS THALIANA ACTIVATING FACTOR, CUP-SHAPED COTYLEDON (NAC) transcription factor SOMBRERO. Surprisingly, the breakdown of the large central vacuole is a relatively late and variable event, preceded by an increase of intracellular calcium levels and acidification, release of mitochondrial matrix proteins, leakage of nuclear and endoplasmic reticulum lumina, and release of fluorescent membrane reporters into the cytosol. In analogy to animal apoptosis, the plasma membrane remains impermeable for proteins during and after PCD execution. Elevated intracellular calcium levels and acidification are sufficient to trigger cell death execution specifically in terminally differentiated root cap cells, suggesting that these ion fluxes act as PCD-triggering signals. This detailed information on the cellular processes occurring during developmental PCD in plants is a pivotal prerequisite for future research into the molecular mechanisms of cell death execution.

Cloning and functional analysis of the BrCUC2 gene in Brassica rapa L.

The CUP-SHAPED COTYLEDON2 (CUC2) gene plays an important role in the formation of apical meristem and organ edges in plants. The apical meristematic tissue of Brassica rapa (B. rapa) is associated with cold resistance, however, the role of the CUC2 gene in cold resistance of B.rapa is unclear. In this study, we used bioinformatics software to analyze the structure of BrCUC2 gene, real-time fluorescence quantitative PCR to detect the expression level of BrCUC2, constructed transgenic Arabidopsis thaliana by the flower dipping method and subcellular localization for functional validation. The results showed that, we isolated a 1104 bp open reading frame of BrCUC2 from the winter B. rapa cultivar 'Longyou 7'. The BrCUC2 contains a highly conserved domain belonging to the NAM superfamily. Its homologus CUC genes contain similar conserved motifs and are closely related to Brassica oleracea (B.oleracea), and the N-terminal of amino acid sequence contains NAC domain. BrCUC2 protein was localized in the nucleus and self-activation tests showed that pGBKT7-BrCUC2 had self-activation. Tissue-specific expression analysis and promoter β-Glucuronidase (GUS) activity showed that BrCUC2 had high expression levels in B. rapa growth points and A. thaliana leaf edges, stems and growth points. After low-temperature stress, BrCUC2 showed greater expression in 'Longyou 7,' which presents strong cold resistance and concave growth points, than in 'Longyou 99,' which presents weak cold resistance and protruding growth points. BrCUC2 promoter contains multiple elements related to stress responses. BrCUC2 overexpression revealed that the phenotype did not differ from that of the wild type during the seedling stage but showed weak growth and a dwarf phenotype during the flowering and mature stages. After low-temperature treatment, the physiological indexes and survival rate of BrCUC2-overexpression lines of Arabidopsis thaliana (A. thaliana) were better than those of the wild type within 12h, although differences were not observed after 24h. These results showed that BrCUC2 improved the low-temperature tolerance of transgenic A. thaliana within a short time. It can provide a foundation for the study of cold resistance in winter B. rapa.

The role of Class II KNOX family in controlling compound leaf patterning in Medicago truncatula.

Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class Ⅱ KNOTTED-like homeobox (KNOXⅡ) genes in the model leguminous plant Medicago truncatula. Phenotypic and genetic analyses suggest that MtKNOX4, 5 are able to repress the leaflet formation, while MtKNOX3, 9, 10 are not involved in this developmental process. Further investigations show that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXI-mediated CK biosynthesis. Additionally, two boundary genes, FUSED COMPOUND LEAF1 (ortholog of Arabidopsis class M KNOX) and NO APICAL MERISTEM (ortholog of Arabidopsis CUP-SHAPED COTYLEDON), are necessary for the MtKNOX4-mediated compound leaf formation. These findings suggest that among the members of MtKNOXII, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves in M. truncatula. This article is protected by copyright. All rights reserved.

Interspersed expression of CUP-SHAPED COTYLEDON2 and REDUCED COMPLEXITY shapes Cardamine hirsuta complex leaf form.

How genetically regulated growth shapes organ form is a key problem in developmental biology. Here, we investigate this problem using the leaflet-bearing complex leaves of Cardamine hirsuta as a model. Leaflet development requires the action of two growth-repressing transcription factors: REDUCED COMPLEXITY (RCO), a homeodomain protein, and CUP-SHAPED COTYLEDON2 (CUC2), a NAC-domain protein. However, how their respective growth-repressive actions are integrated in space and time to generate complex leaf forms remains unknown. By using live imaging, we show that CUC2 and RCO are expressed in an interspersed fashion along the leaf margin, creating a distinctive striped pattern. We find that this pattern is functionally important because forcing RCO expression in the CUC2 domain disrupts auxin-based marginal patterning and can abolish leaflet formation. By combining genetic perturbations with time-lapse imaging and cellular growth quantifications, we provide evidence that RCO-mediated growth repression occurs after auxin-based leaflet patterning and in association with the repression of cell proliferation. Additionally, through the use of genetic mosaics, we show that RCO is sufficient to repress both cellular growth and proliferation in a cell-autonomous manner. This mechanism of growth repression is different to that of CUC2, which occurs in proliferating cells. Our findings clarify how the two growth repressors RCO and CUC2 coordinate to subdivide developing leaf primordia into distinct leaflets and generate the complex leaf form. They also indicate different relationships between growth repression and cell proliferation in the patterning and post-patterning stages of organogenesis.