Transcription factor RcNAC091 enhances rose drought tolerance through the abscisic acid-dependent pathway.

NAC (NAM, ATAF1,2, and CUC2) transcription factors (TFs) play critical roles in controlling plant growth, development, and abiotic stress responses. However, few studies have examined NAC proteins related to drought stress tolerance in rose (Rosa chinensis). Here, we identified a drought- and abscisic acid (ABA)-induced NAC TF, RcNAC091, that localizes to the nucleus and has transcriptional activation activity. Virus-induced silencing of RcNAC091 resulted in decreased drought stress tolerance, and RcNAC091 overexpression had the opposite effect. Specifically, ABA mediated RcNAC091-regulated drought tolerance. A transcriptomic comparison showed altered expression of genes involved in ABA signaling and oxidase metabolism in RcNAC091-silenced plants. We further confirmed that RcNAC091 directly targets the promoter of RcWRKY71 in vivo and in vitro. Moreover, RcWRKY71-slienced rose plants were not sensitive to both ABA and drought stress, whereas RcWRKY71-overexpressing plants were hypersensitive to ABA, which resulted in drought-tolerant phenotypes. The expression of ABA biosynthesis- and signaling-related genes was impaired in RcWRKY71-slienced plants, suggesting that RcWRKY71 might facilitate the ABA-dependent pathway. Therefore, our results show that RcWRKY71 is transcriptionally activated by RcNAC091, which positively modulates ABA signaling and drought responses. The results of this study provide insights into the roles of TFs as functional links between RcNAC091 and RcWRKY71 in priming resistance; our findings also have implications for the approaches to enhance the drought resistance of roses.

ANAC102 predominantly expresses a nuclear protein and acts as a negative regulator of methyl viologen-induced retrograde signaling.

Plants, being sessile organisms, constantly need to respond to environmental stresses, often leading to the accumulation of reactive oxygen species (ROS). While ROS can be harmful, they also act as messengers guiding plant growth and stress responses. Because chloroplasts are sensitive to environmental changes and are both a source and target of ROS during stress conditions, they are important in conveying environmental changes to the nucleus, where acclimation responses are coordinated to maintain organellar and overall cellular homeostasis. ANAC102 has previously been established as a regulator of ß-cyclocitral-mediated chloroplast-to-nucleus signaling, protecting plants against photooxidative stress. However, debates persist about where ANAC102 is located - in chloroplasts or in the nucleus. Our study, utilizing the genomic ANAC102 sequence driven by its native promoter, establishes ANAC102 primarily as a nuclear protein, lacking a complete N-terminal chloroplast-targeting peptide. Moreover, our research reveals the sensitivity of plants overexpressing ANAC102 to severe superoxide-induced chloroplast oxidative stress. Transcriptome analysis unraveled ANAC102's dual role in negatively and positively regulating genome-wide transcriptional responses to chloroplast oxidative stress. Through the integration of published data and our own study, we constructed a comprehensive transcriptional network, which suggests that ANAC102 exerts direct and indirect control over transcriptional responses through downstream transcription factor networks, providing deeper insights into the ANAC102-mediated regulatory landscape during oxidative stress.

Genome-wide analysis of the rose (Rosa chinensis) NAC family and characterization of RcNAC091.

KEY MESSAGE: A genome-wide analysis identified 116 NAC genes in rose, including stress-related ones with different expression patterns under drought and salt stress. Silencing of RcNAC091, a member of the ATAF subfamily, decreased dehydration tolerance in rose. The NAC (NAM, ATAF, and CUC) transcription factors (TFs) are plant-specific proteins that regulate various developmental processes and stress responses. However, knowledge of the NAC TFs in rose (Rosa chinensis), one of the most important horticultural crops, is limited. In this study, 116 NAC genes were identified from the rose genome and classified into 16 subfamilies based on protein phylogenetic analysis. Chromosomal mapping revealed that the RcNAC genes were unevenly distributed on the seven chromosomes of rose. Gene structure and motif analysis identified a total of ten conserved motifs, of which motifs 1-7 were highly conserved and present in most rose NACs, while motifs 8-10 were present only in a few subfamilies. Further study of the stress-related RcNACs based on the transcriptome data showed differences in the expression patterns among the organs, at various floral developmental stages, and under drought and salt stress in rose leaves and roots. The stress-related RcNACs possessed cis-regulatory elements (CREs) categorized into three groups corresponding to plant growth and development, phytohormone response, and abiotic and biotic stress response. Reverse transcription-quantitative real-time PCR (RT-qPCR) analysis of 11 representative RcNACs revealed their differential expression in rose leaves and roots under abscisic acid (ABA), polyethylene glycol (PEG), and sodium chloride (NaCl) treatments. Furthermore, the silencing of RcNAC091 verified its role in positively regulating the dehydration stress response. Overall, the present study provides valuable insights into stress-related RcNACs and paves the way for stress tolerance in rose.

Genome-wide analysis of BURP genes and identification of a BURP-V gene RcBURP4 in Rosa chinensis.

KEY MESSAGE: Nine RcBURPs have been identified in Rosa chinensis, and overexpression of RcBURP4 increased ABA, NaCl sensitivity, and drought tolerance in transgenic Arabidopsis. BURP proteins are unique to plants and may contribute greatly to growth, development, and stress responses of plants. Despite the vital role of BURP proteins, little is known about these proteins in rose (Rosa spp.). In the present study, nine genes belonging to the BURP family in R. chinensis were identified using multiple bioinformatic approaches against the rose genome database. The nine RcBURPs, with diverse structures, were located on all chromosomes of the rose genome, except for Chr2 and Chr3. Phylogenic analysis revealed that these RcBURPs can be classified into eight subfamilies, including BNM2-like, PG1ß-like, USP-like, RD22-like, BURP-V, BURP-VI, BURP-VII, and BURP-VIII. Conserved motif and exon-intron analyses indicated a conserved pattern within the same subfamily. The presumed cis-regulatory elements (CREs) within the promoter region of each RcBURP were analyzed and the results showed that all RcBURPs contained different types of CREs, including abiotic stress-, light response-, phytohormones response-, and plant growth and development-related CREs. Transcriptomic analysis revealed that a BURP-V member, RcBURP4, was induced in rose leaves and roots under mild and severe drought treatments. We then overexpressed RcBURP4 in Arabidopsis and examined its role under abscisic acid (ABA), NaCl, polyethylene glycol (PEG), and drought treatments. Nine stress-responsive genes expression were changed in RcBURP4-overexpressing leaves and roots. Furthermore, RcBURP4-silenced rose plants exhibited decreased tolerance to dehydration. The results obtained from this study provide the first comprehensive overview of RcBURPs and highlight the importance of RcBURP4 in rose plant.

Whole-genome characterization of Rosa chinensis AP2/ERF transcription factors and analysis of negative regulator RcDREB2B in Arabidopsis.

BACKGROUND: Rose (Rosa chinensis) is a traditional famous flower with valuable ornamental characteristics. However, drought stress restricts its growth and development, leading to an abnormal phenotype. One of the main transcription factor (TF) protein groups in the plant kingdom are the APETALA2/ethylene-responsive factor (AP2/ERF) proteins and are potentially involved in the growth and stress responses of various plants. RESULTS: Our investigation mainly focused on exploring the genome of rose and thereby we discovered 135 apparent AP2/ERF TFs. Phylogenic analyses revealed that RcAP2/ERF genes are categorized into DREB, Soloist, AP2, and ERF subfamilies, and are further classified these into 17 groups, with the same as Malus domestica and Arabidopsis thaliana. The analysis of the gene structure revealed that the introns ranged from 0 to 9 in number. Pattern examination demonstrated that the RcAP2/ERF predominantly consists of typical AP2 domains, of which the 2nd motif is the most ubiquitous. Distributions of cis-acting elements indicated that members of the AP2/ERF family are frequently involved in growth and development, phytohormone and stress response in rose species. Also, the distribution mapping of the rose chromosomes indicated that AP2/ERF class genes are dispersed among all seven chromosomes. Additionally, we isolated a novel DREB A2 subgroup gene and named it RcDREB2B. Subsequently, the RcDREB2B transcript accumulation was repressed under the mild and severe drought stress in the root samples of rose. RcDREB2B was targeted to the nucleus and exhibited transactivation in yeast cells. The overexpression of RcDREB2B was found to promote sensitivity to a higher salt concentration, ABA, and PEG at the germination and post-germination stages. Twelve putative osmotic and ABA-related genes were impaired in RcDREB2B-overexpressing plants. CONCLUSIONS: The results provide comprehensive information regarding the gene structure, phylogenic, and distribution of the rose AP2/ERF family and bring insight into the complex transcriptional gene regulation of RcAP2/ERF. Findings in this study would also contribute to further understanding of the RcDREB2B gene in rose.

Physiological Characteristic Changes and Full-Length Transcriptome of Rose (Rosa chinensis) Roots and Leaves in Response to Drought Stress.

Rose (Rosa chinensis) is the most important ornamental crops worldwide. However, the physiological and molecular mechanism of rose under drought stress remains elusive. In this study, we analyzed the changes of photosynthetic and phytohormone levels in the leaves and roots of rose seedlings grown under control (no drought), mild drought (MD) and severe drought stress. The total chlorophyll content and water use efficiency were significantly enhanced under MD in rose leaves. In addition, the concentration of ABA was higher in the leaves compared to the roots, whereas the roots accumulated more IAA, methylindole-3-acetic acid and indole-3-propionic acid. We also constructed the first full-length transcriptome for rose, and identified 96,201,862 full-length reads of average length 1,149 bp that included 65,789 novel transcripts. A total of 3,657 and 4,341 differentially expressed genes (DEGs) were identified in rose leaves and roots respectively. KEGG pathway analysis showed enrichment of plant hormone, signal transduction and photosynthesis are among the DEGs. 42,544 alternatively spliced isoforms were also identified, and alternative 3' splice site was the major alternative splicing (AS) event among the DEGs. Variations in the AS patterns of three genes between leaves and roots indicated the possibility of tissue-specific posttranscriptional regulation in response to drought stress. Furthermore, 2,410 novel long non-coding RNAs were detected that may participate in regulating the drought-induced DEGs. Our findings identified previously unknown splice sites and new genes in the rose transcriptome, and elucidated the drought stress-responsive genes as well as their intricate regulatory networks.

Analysis of the thaumatin-like genes of Rosa chinensis and functional analysis of the role of RcTLP6 in salt stress tolerance.

MAIN CONCLUSION: A total of 27 rose thaumatin-like protein (TLP) genes were identified from the rose genome through bioinformatics analyses. RcTLP6 was found to confer salinity stress tolerance in rose. Thaumatin-like proteins (TLPs) play critical roles in regulating many biological processes, including abiotic and biotic stress responses in plants. Here, we conducted a genome-wide screen of TLPs in rose (Rosa chinensis) and identified 27 RcTLPs. The identified RcTLPs, as well as other TLPs from six different plant species, were placed into nine groups based on a phylogenetic analysis. An analysis of the intron-exon structures of the TLPs revealed a high degree of similarity. RcTLP genes were found on all chromosomes except for chromosome four. Cis-regulatory elements (CEs) were identified in the promoters of all RcTLPs, including CEs associated with growth, development and hormone-responsiveness, as well as abiotic and biotic responses, indicating they play diverse roles in rose. Transcriptomics analysis revealed that RcTLPs had tissue-specific expression patterns, and several root-preferential RcTLPs were responsive to drought and salinity stress. Quantitative PCR analysis of six RcTLPs under ABA, PEG and NaCl treatment confirmed the differentially expressed genes identified in the transcriptomics experiment. In addition, silencing RcTLP6 in rose leaves led to decreased tolerance to salinity stress. We also screened proteins which may interact with RcTLP6 to understand its biological roles. This study represents the first report of the TLP gene family in rose and expands the current understanding of the role that RcTLP6 plays in salt tolerance. These findings lay a foundation for future utilization of RcTLPs to improve rose abiotic stress tolerance.

Chlorogenic acid inhibits osteosarcoma carcinogenesis via suppressing the STAT3/Snail pathway.

Chlorogenic acid (CA) is a polyphenol compound that possesses anticancer effects on several types of tumors. However, there are few previous studies concerning the protective effects of CA on osteosarcoma. The current study aimed to examine the toxicity of CA to osteosarcoma cells and to explore the potential mechanisms. Cell growth was evaluated using cell counting kit-8 assay and Western blot analysis of proliferating cell nuclear antigen (PCNA). Apoptosis was assessed by flow cytometry analysis using flow cytometry and caspase-3/7 activity assay. The expression changes of the signal transducer and activator of transcription 3 (STAT3)/Snail pathway were detected by Western blot analysis. We found that CA dose-dependently inhibited cell viability and PCNA expression in osteosarcoma cells. Meanwhile, CA treatment increased the apoptotic rate and caspase-3/7 activity in osteosarcoma cells in a concentration-dependent manner. We found that CA concentration-dependently inhibited the activation of the STAT3/Snail pathway in osteosarcoma cells. Inhibition of the STAT3/Snail pathway by si-STAT3 retarded the growth and induced apoptosis of osteosarcoma cells. Mechanistically, activation of the STAT3/Snail pathway by pcDNA-STAT3 reversed the effects of CA on osteosarcoma cell growth and apoptosis. In conclusion, CA inhibited osteosarcoma carcinogenesis by suppressing osteosarcoma cell growth and inducing apoptosis, which was involved in inactivation of the STAT3/Snail pathway. Therefore, our study suggested that CA might have good therapy prospects in osteosarcoma therapy.

A RhABF2/Ferritin module affects rose (Rosa hybrida) petal dehydration tolerance and senescence by modulating iron levels.

Plants often develop the capacity to tolerate moderate and reversible environmental stresses, such as drought, and to re-establish normal development once the stress has been removed. An example of this phenomenon is provided by cut rose (Rosa hybrida) flowers, which experience typical reversible dehydration stresses during post-harvest handling after harvesting at the bud stages. The molecular mechanisms involved in rose flower dehydration tolerance are not known, however. Here, we characterized a dehydration- and abscisic acid (ABA)-induced ferritin gene (RhFer1). Dehydration-induced free ferrous iron (Fe2+ ) is preferentially sequestered by RhFer1 and not transported outside of the petal cells, to restrict oxidative stresses during dehydration. Free Fe2+ accumulation resulted in more serious oxidative stresses and the induction of genes encoding antioxidant enzyme in RhFer1-silenced petals, and poorer dehydration tolerance was observed compared with tobacco rattle virus (TRV) controls. We also determined that RhABF2, an AREB/ABF transcription factor involved in the ABA signaling pathway, can activate RhFer1 expression by directly binding to its promoter. The silencing of RhABF2 decreased dehydration tolerance and disrupted Fe homeostasis in rose petals during dehydration, as did the silencing of RhFer1. Although both RhFer1 and Fe transporter genes are induced during flower natural senescence in plants, the silencing of RhABF2 or RhFer1 accelerates the petal senescence processes. These results suggest that the regulatory module RhABF2/RhFer1 contributes to the maintenance of Fe levels and enhances dehydration tolerance through the action of RhFer1 locally sequestering free Fe2+ under dehydration conditions, and plays synergistic roles with transporter genes during flower senescence.

RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa hybrida) petal senescence.

Rose (Rosa hybrida) is one of the most important ornamental plants worldwide; however, senescence of its petals terminates the ornamental value of the flower, resulting in major economic loss. It is known that the hormones abscisic acid (ABA) and ethylene promote petal senescence, while gibberellins (GAs) delay the process. However, the molecular mechanisms underlying the antagonistic effects amongst plant hormones during petal senescence are still unclear. Here we isolated RhHB1, a homeodomain-leucine zipper I transcription factor gene, from rose flowers. Quantitative RT-PCR and GUS reporter analyses showed that RhHB1 was strongly expressed in senescing petals, and its expression was induced by ABA or ethylene in petals. ABA or ethylene treatment clearly accelerated rose petal senescence, while application of the gibberellin GA3 delayed the process. However, silencing of RhHB1 delayed the ABA- or ethylene-mediated senescence, and resulted in higher petal anthocyanin levels and lower expression of RhSAG12. Moreover, treatment with paclobutrazol, an inhibitor of GA biosynthesis, repressed these delays. In addition, silencing of RhHB1 blocked the ABA- or ethylene-induced reduction in expression of the GA20 oxidase encoded by RhGA20ox1, a gene in the GA biosynthetic pathway. Furthermore, RhHB1 directly binds to the RhGA20ox1 promoter, and silencing of RhGA20ox1 promoted petal senescence. Eight senescence-related genes showed substantial differences in expression in petals after treatment with GA3 or paclobutrazol. These results suggest that RhHB1 mediates the antagonistic effect of GAs on ABA and ethylene during rose petal senescence, and that the promotion of petal senescence by ABA or ethylene operates through an RhHB1-RhGA20ox1 regulatory checkpoint.

RhNAC3, a stress-associated NAC transcription factor, has a role in dehydration tolerance through regulating osmotic stress-related genes in rose petals.

Petal cell expansion depends on cell wall metabolism, changes in cell turgor pressure and restructuring of the cytoskeleton, and recovery ability of petal cell expansion is defined as an indicator of dehydration tolerance in flowers. We previously reported that RhNAC2, a development-related NAC domain transcription factor, confers dehydration tolerance through regulating cell wall-related genes in rose petals. Here, we identify RhNAC3, a novel rose SNAC gene, and its expression in petals induced by dehydration, wounding, exogenous ethylene and abscisic acid (ABA). Expression studies in Arabidopsis protoplasts and yeast show that RhNAC3 has transactivation activity along its full length and in the carboxyl-terminal domain. Silencing RhNAC3 in rose petals by virus-induced gene silencing (VIGS) significantly decreased the cell expansion of rose petals under rehydration conditions. In total, 24 of 27 osmotic stress-related genes were down-regulated in RhNAC3-silenced rose petals, while only 4 of 22 cell expansion-related genes were down-regulated. Overexpression of RhNAC3 in Arabidopsis gave improved drought tolerance, with lower water loss of leaves in transgenic plants. Arabidopsis ATH1 microarray analysis showed that RhNAC3 regulated the expression of stress-responsive genes in overexpressing lines, and further analysis revealed that most of the RhNAC3-up-regulated genes were involved in the response to osmotic stress. Comparative analysis revealed that different transcription regulation existed between RhNAC3 and RhNAC2. Taken together, these data indicate that RhNAC3, as a positive regulator, confers dehydration tolerance of rose petals mainly through regulating osmotic adjustment-associated genes.

Genome-Wide Identification, Characterization, and Expression Analysis of the HD-Zip Gene Family in Lagerstroemia for Regulating Plant Height.

The Homeodomain leucine zipper (HD-Zip) family of transcription factors is crucial in helping plants adapt to environmental changes and promoting their growth and development. Despite research on the HD-Zip family in various plants, studies in Lagerstroemia (crape myrtle) have not been reported. This study aimed to address this gap by comprehensively analyzing the HD-Zip gene family in crape myrtle. This study identified 52 HD-Zip genes in the genome of Lagerstroemia indica, designated as LinHDZ1-LinHDZ52. These genes were distributed across 22 chromosomes and grouped into 4 clusters (HD-Zip I-IV) based on their phylogenetic relationships. Most gene structures and motifs within each cluster were conserved. Analysis of protein properties, gene structure, conserved motifs, and cis-acting regulatory elements revealed diverse roles of LinHDZs in various biological contexts. Examining the expression patterns of these 52 genes in 6 tissues (shoot apical meristem, tender shoot, and mature shoot) of non-dwarf and dwarf crape myrtles revealed that 2 LinHDZs (LinHDZ24 and LinHDZ14) and 2 LinHDZs (LinHDZ9 and LinHDZ35) were respectively upregulated in tender shoot of non-dwarf crape myrtles and tender and mature shoots of dwarf crape myrtles, which suggested the important roles of these genes in regulate the shoot development of Lagerstroemia. In addition, the expression levels of 2 LinHDZs (LinHDZ23 and LinHDZ34) were significantly upregulated in the shoot apical meristem of non-dwarf crape myrtle. These genes were identified as key candidates for regulating Lagerstroemia plant height. This study enhanced the understanding of the functions of HD-Zip family members in the growth and development processes of woody plants and provided a theoretical basis for further studies on the molecular mechanisms underlying Lagerstroemia plant height.

RcMYB8 enhances salt and drought tolerance in rose (Rosa chinensis) by modulating RcPR5/1 and RcP5CS1.

Plant Myeloblastosis (MYB) proteins function crucially roles upon variegated abiotic stresses. Nonetheless, their effects and mechanisms in rose (Rosa chinensis) are not fully clarified. In this study, we characterized the effects of rose RcMYB8 under salt and drought tolerances. For induction of the RcMYB8 expression, NaCl and drought stress treatment were adopted. Rose plants overexpressing RcMYB8 displayed enhanced tolerance to salinity and drought stress, while silencing RcMYB8 resulted in decreased tolerance, as evidenced by lowered intra-leaf electrolyte leakage and callose deposition, as well as photosynthetic sustainment under stressed conditions. Here, we further show that RcMYB8 binds similarly to the promoters of RcPR5/1 and RcP5C51 in vivo and in vitro. Inhibiting RcP5CS1 by virus-induced gene silencing led to decreased drought tolerance through the reactive oxygen species (ROS) homeostatic regulation. RcP5CS1-silenced plants showed an increase in ion leakage and reduce of proline content, together with the content of malondialdehyde (MDA) increased, lowered activities of Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Our study highlights the transcriptional modulator role of RcMYB8 in drought and salinity tolerances, which bridges RcPR5/1 and RcP5CS1 by promoting ROS scavenging. Besides, it is probably applicable to the rose plant engineering for enhancing their abiotic stress tolerances.

RhEXPA4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis.

Drought and high salinity are major environmental conditions limiting plant growth and development. Expansin is a cell-wall-loosening protein known to disrupt hydrogen bonds between xyloglucan and cellulose microfibrils. The expression of expansin increases in plants under various abiotic stresses, and plays an important role in adaptation to these stresses. We aimed to investigate the role of the RhEXPA4, a rose expansin gene, in response to abiotic stresses through its overexpression analysis in Arabidopsis. In transgenic Arabidopsis harboring the Pro RhEXPA4 ::GUS construct, RhEXPA4 promoter activity was induced by abscisic acid (ABA), drought and salt, particularly in zones of active growth. Transgenic lines with higher RhEXPA4 level developed compact phenotypes with shorter stems, curly leaves and compact inflorescences, while the lines with relatively lower RhEXPA4 expression showed normal phenotypes, similar to the wild type (WT). The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under salt stress and ABA treatments. Transgenic plants showed enhanced tolerance to drought and salt stresses: they displayed higher survival rates after drought, and exhibited more lateral roots and higher content of leaf chlorophyll a under salt stress. Moreover, high-level RhEXPA4 overexpressors have multiple modifications in leaf blade epidermal structure, such as smaller, compact cells, fewer stomata and midvein vascular patterning in leaves, which provides them with more tolerance to abiotic stresses compared to mild overexpressors and the WT. Collectively, our results suggest that RhEXPA4, a cell-wall-loosening protein, confers tolerance to abiotic stresses through modifying cell expansion and plant development in Arabidopsis.

RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose petals.

Dehydration inhibits petal expansion resulting in abnormal flower opening and results in quality loss during the marketing of cut flowers. We constructed a suppression subtractive hybridization library from rose (Rosa hybrida) flowers containing 3,513 unique expressed sequence tags and analyzed their expression profiles during cycles of dehydration. We found that 54 genes were up-regulated by the first dehydration, restored or even down-regulated by rehydration, and once again up-regulated by the second dehydration. Among them, we identified a putative NAC family transcription factor (RhNAC2). With transactivation activity of its carboxyl-terminal domain in yeast (Saccharomyces cerevisiae) cell and Arabidopsis (Arabidopsis thaliana) protoplast, RhNAC2 belongs to the NAC transcription factor clade related to plant development in Arabidopsis. A putative expansin gene named RhEXPA4 was also dramatically up-regulated by dehydration. Silencing RhNAC2 or RhEXPA4 in rose petals by virus-induced gene silencing significantly decreased the recovery of intact petals and petal discs during rehydration. Overexpression of RhNAC2 or RhEXPA4 in Arabidopsis conferred strong drought tolerance in the transgenic plants. RhEXPA4 expression was repressed in RhNAC2-silenced rose petals, and the amino-terminal binding domain of RhNAC2 bound to the RhEXPA4 promoter. Twenty cell wall-related genes, including seven expansin family members, were up-regulated in Arabidopsis plants overexpressing RhNAC2. These data indicate that RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose petals and that RhEXPA4 expression may be regulated by RhNAC2.

RcbHLH59-RcPRs module enhances salinity stress tolerance by balancing Na+/K+ through callose deposition in rose (Rosa chinensis).

Basic helix-loop-helix (bHLH) proteins play pivotal roles in plant growth, development, and stress responses. However, the molecular and functional properties of bHLHs have not been fully characterized. In this study, a novel XI subgroup of the bHLH protein gene RcbHLH59 was isolated and identified in rose (Rosa sp.). This gene was induced by salinity stress in both rose leaves and roots, and functioned as a transactivator. Accordingly, silencing RcbHLH59 affected the antioxidant system, Na +/K + balance, and photosynthetic system, thereby reducing salt tolerance, while the transient overexpression of RcbHLH59 improved salinity stress tolerance. Additionally, RcbLHLH59 was found to regulate the expression of sets of pathogenesis-related (PR) genes in RcbHLH59-silenced (TRV-RcbHLH59) and RcbHLH59-overexpressing (RcbHLH59-OE) rose plants. The RcPR4/1 and RcPR5/1 transcript levels showed opposite changes in the TRV-RcbHLH59 and RcbHLH59-OE lines, suggesting that these two genes are regulated by RcbHLH59. Further analysis revealed that RcbHLH59 binds to the promoters of RcPR4/1 and RcPR5/1, and that the silencing of RcPR4/1 or RcPR5/1 led to decreased tolerance to salinity stress. Moreover, callose degradation- and deposition-related genes were impaired in RcPR4/1- or RcPR5/1-silenced plants, which displayed a salt tolerance phenotype by balancing the Na+/K+ ratio through callose deposition. Collectively, our data highlight a new RcbLHLH59-RcPRs module that positively regulates salinity stress tolerance by balancing Na+/K+ and through callose deposition in rose plants.

Multiplex PCR for 17 Y-chromosome Specific Short Tandem Repeats (STR) to enhance the reliability of fetal sex determination in maternal plasma.

The aim of the study was to demonstrate the influence of target gene and amplification product length on the performance of fetal gender determination systems using maternal plasma. A total of 40 pairs of plasma DNA samples from pregnant women and genomic DNA samples from maternal blood, amniotic fluid and paternal blood were isolated for gender determination by amplification of the amelogenin gene and 17 Y-chromosome STR loci, using three different commercial kits. The gender of the fetuses was confirmed by cytogenetic analysis or phenotype at birth. Both the AmpFℓSTR-Identifiler amplification kit and the Mini-STR Amplification kit for amelogenin gene detection were reliable in determining fetal gender (92.0% and 96.0%, respectively), but false negatives were present in both systems. AmpFℓSTR-Yfiler was found to be fully reliable as it amplified Y-STR in all cases of pregnancies with male fetuses and thus was 100% correct in determining fetal gender. The results demonstrated that multiple fluorescent PCR for 17 Y-STR loci was more reliable than AMELY gene testing in fetal sex determination with maternal plasma. We also found that the shorter amplification products could improve the performance of fetal gender determination systems.

Overexpression of LtKNOX1 from Lilium tsingtauense in Nicotiana benthamiana affects the development of leaf morphology.

Leaves are the main vegetative organs of the aboveground part of plants and play an important role in plant morphogenesis. KNOTTED-LIKE HOMEOBOX (KNOX) plays a crucial role in regulating leaf cell fate and maintaining leaf development. In this study, we analyzed LtKNOX1 from Lilium tsingtauense and illustrated its function in transgenic plants. Tissue-specific expression analysis indicated that LtKNOX1 was highly expressed in stems, young flower buds, and shoot apical meristems (SAMs). Ectopic overexpression of LtKNOX1 in Nicotiana benthamiana suggested that transformants with mild phenotypes were characterized by foliar wrinkles and mildly curled leaves; transformants with intermediate phenotypes showed severely crimped blades and narrow leaf angles, and the most severe phenotypes lacked normal SAMs and leaves. Moreover, the expression levels of genes involved in the regulation of KNOX in transgenic plants were detected, including ASYMMETRIC LEAVES1, PIN-FORMED 1, GA20-oxidase, CUP-SHAPED COTYLEDON 2, CLAVATA 1 and WUSCHEL(WUS), and the expression of other genes were down-regulated except WUS. This study contributes to our understanding of the LtKNOX1 function.

Genome-wide identification and expression analysis of anaphase promoting complex/cyclosome (APC/C) in rose.

The anaphase promoting complex/cyclosome (APC/C) is a large multi-subunit complex, regulating plant development and cell cycle. In plants, the APC/C gene family has been identified in Arabidopsis, rice, and maize. The APC/Cs in rose has not yet been reported. In this study, a total of 19 APC/C genes were identified in rose. Furthermore, we also investigated phylogenetic relationships, chromosomal distribution, gene structure, motif analysis, promoter sequence analysis and expression pattern of RhAPC/C genes. Synteny analysis indicated that AtAPC/Cs and RhAPC/Cs show a high degree of conservation. RhAPC/C promoters contains numerous cis-elements involved in plant morphogenesis, hormone response and stress response. Based on the transcription of RhAPC/Cs in different tissues and developmental stages, it appears that RhAPC/Cs may play a variety of roles in rose growth and development. RhAPC/Cs have limitations in the time and space during which they respond to hormones and abiotic stress. RhAPC5, RhAPC11d, RhAPC13a and RhAPC13c may play a role in rose responding to abiotic stress. The expression of RhAPC10 was altered by infection with fungal pathogen. Our study will serve as a basis for determining the functional role of APC/C genes in roses and help future research on woody plants.

De novo transcriptomic analysis and identification of EST-SSR markers in Stephanandra incisa.

Stephanandra incisa is a wild-type shrub with beautiful leaves and white flowers and is commonly used as a garden decoration accessory. However, the limited availability of genomic data of S. incisa has restricted its breeding process. Here, we identified EST-SSR markers using de novo transcriptome sequencing. In this study, a transcriptome database containing 35,251 unigenes, having an average length of 985 bp, was obtained from S. incisa. From these unigene sequences, we identified 5,555 EST-SSRs, with a distribution density of one SSR per 1.60 kb. Dinucleotides (52.96%) were the most detected SSRs, followed by trinucleotides (34.64%). From the EST-SSR loci, we randomly selected 100 sites for designing primer and used the DNA of 60 samples to verify the polymorphism. The average value of the effective number of alleles (Ne), Shannon's information index (I), and expective heterozygosity (He) was 1.969, 0.728, and 0.434, respectively. The polymorphism information content (PIC) value was in the range of 0.108 to 0.669, averaging 0.406, which represented a middle polymorphism level. Cluster analysis of S. incisa were also performed based on the obtained EST-SSR data in our work. As shown by structure analysis, 60 individuals could be classified into two groups. Thus, the identification of these novel EST-SSR markers provided valuable sequence information for analyzing the population structure, genetic diversity, and genetic resource assessment of S. incisa and other related species.