Comprehensive analysis of AHL genes in Malus domestica reveals the critical role of MdAHL6 in flowering induction.

AT-hook motif nuclear localized (AHL) genes are crucial in various biological processes, yet the AHL gene family in apples has remained largely unexplored. In this study, we isolated 36 MdAHL genes from the apple genome and grouped them into two distinct clades. We characterized the gene structure, conserved motifs, protein biochemical properties, and promoter regions of the MdAHL genes. Transcriptional analysis revealed that MdAHL genes are preferentially and predominantly expressed in flowers and leaves. Notably, during the floral induction phase, the MdAHL6 gene exhibited remarkably high transcriptional activity. Overexpression of MdAHL6 resulted in shortened hypocotyls and delayed flowering by regulating hypocotyl- and floral-related genes. Y1H, EMSA, GUS activity, and molecular docking assays revealed that MdAHL6 directly binds to AT-rich regions, inhibiting the expression of FLOWERING LOCUS T (MdFT). Furthermore, Y2H, pull-down, and BiFC assays demonstrated a physical interaction between MdAHL6 and the class II knotted-like transcription factor MdKNOX19, which significantly enhances the inhibitory effect of MdAHL6 on MdFT expression. This comprehensive initial analysis unveils the critical role of the MdKNOX19-MdAHL6-MdFT module in flowering induction and lays a theoretical foundation for future functional exploration.

The CaABCG14 transporter gene regulates the capsaicin accumulation in Pepper septum.

Capsaicin (CAP), a crucial compound found in chili peppers, not only contributes to their spicy flavor but also possesses several industrial applications. CAP biosynthetic pathway is well known, while its transport mechanism remains elusive. Herein, we performed a comparative transcriptome analysis conducted on pepper fruit tissues at three different stages of development. Four important CAP transporter genes, including one MATE and three ABCs, were identified by differential expression and WGCNA analysis. Specifically, the expression patterns of three ABC genes were assessed in the septum of fruits from nine distinct genotypes of peppers with high capsaicin levels. Interestingly, CaABCG14 was associated with variations in CAP concentration and co-expressed with genes involved in CAP biosynthesis. Transient expression assay revealed that CaABCG14 is localized to the membrane and nucleus. Silencing of CaABCG14 resulted in a notable reduction in the levels of CAP contents and the expression of its biosynthetic genes in the septum of pepper. The overexpression of CaABCG14 greatly intensified the cytotoxic effects of CAP on the yeast cells. Taken together, we for the first time identified a new transporter gene CaABCG14, regulating the CAP accumulation in pepper septum. These findings offer a fresh molecular theoretical framework for CAP transport and accumulation.

Changes in hemicellulose metabolism in banana peel during fruit development and ripening.

Hemicellulose is key in determining the fate of plant cell wall in almost all growth and developmental stages. Nevertheless, there is limited knowledge regarding its involvement in the development and ripening of banana fruit. This study investigated changes in the temporal-spatial distribution of various hemicellulose components, hemicellulose content, activities of the main hydrolysis enzymes, and transcription level of the main hemicellulose-related gene families in banana peels. Both hemicellulose and xylan contents were positively correlated to the fruit firmness observed in our previous study. On the contrary, the xylanase activity was negatively correlated to xylan content and the fruit firmness. The vascular bundle cells, phloem, and cortex of bananas are abundant in xyloglucan, xylan, and mannan contents. Interestingly, the changes in the signal intensity of the CCRC-M104 antibody recognizing non-XXXG type xyloglucan are positively correlated to hemicellulose content. According to RNA-Seq analysis, xyloglucan and xylan-related genes were highly active in the early stages of growth, and the expression of MaMANs and MaXYNs increased as the fruit ripened. The abundance of plant hormonal and growth-responsive cis-acting elements was detected in the 2 kb upstream region of hemicellulose-related gene families. Interaction between hemicellulose and cell wall-specific proteins and MaKCBP1/2, MaCKG1, and MaHKL1 was found. The findings shed light on cell wall hemicellulose's role in banana fruit development and ripening, which could improve nutrition, flavor, and reduce postharvest fruit losses.

Cucumber JASMONATE ZIM-DOMAIN 6 interaction with transcription factor MYB6 impairs waterlogging-triggered adventitious rooting.

Waterlogging is a serious abiotic stress that drastically decreases crop productivity by damaging the root system. Jasmonic acid (JA) inhibits waterlogging-induced adventitious root (AR) formation in cucumber (Cucumis sativus L.). However, we still lack a profound mechanistic understanding of how JA governs AR formation under waterlogging stress. JAZ (JASMONATE ZIM-DOMAIN) proteins are responsible for repressing JA signaling in a transcriptional manner. In this study, we showed that overexpressing CsJAZ8 inhibited the formation of ARs triggered by waterlogging. Molecular analyses revealed that CsJAZ8 inhibited the activation of the R2R3-MYB transcription factor CsMYB6 via direct interaction. Additionally, silencing of CsMYB6 negatively impacted AR formation under waterlogging stress, as CsMYB6 could directly bind to the promoters of 1-aminocyclopropane-1-carboxylate oxidase2 gene CsACO2 and gibberellin 20-oxidases gene CsGA20ox2, facilitating the transcription of these genes. The overexpression of CsACO2 and CsGA20ox2 led to increased levels of ethylene and gibberellin, which facilitated AR formation under waterlogging conditions. On the contrary, silencing these genes resulted in contrasting phenotypes of AR formation. These results highlight that the transcriptional cascade of CsJAZ8 and CsMYB6 plays a critical role in regulating hormonal-mediated cucumber waterlogging-triggered AR formation by inhibiting ethylene and gibberellin accumulation. We anticipate that our findings will provide insights into the molecular mechanisms that drive the emergence of AR in cucumber plants under waterlogging stress.

Reviving resilience: MEcPP-mediated ASK1-IMPα-9-TRP2 stress-responsive module.

Chloroplasts are not only critical photosynthesis sites in plants, but they also participate in plastidial retrograde signaling in response to developmental and environmental signals. MEcPP (2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate) is an intermediary in the methylerythritol phosphate (MEP) pathway in chloroplasts. It is a critical precursor for the synthesis of isoprenoids and terpenoid derivatives, which play crucial roles in plant growth and development, photosynthesis, reproduction, and defense against environmental constraints. Accumulation of MEcPP under stressful conditions triggers the expression of IMPα-9 and TPR2, contributing to the activation of abiotic stress-responsive genes. In this correspondence, we discuss plastidial retrograde signaling in support of a recently published paper in Molecular Plant (Zeng et al. 2024). We hope that it can shed more insight on the retrograde signaling cascade.

Surviving a Double-Edged Sword: Response of Horticultural Crops to Multiple Abiotic Stressors.

Climate change-induced weather events, such as extreme temperatures, prolonged drought spells, or flooding, pose an enormous risk to crop productivity. Studies on the implications of multiple stresses may vary from those on a single stress. Usually, these stresses coincide, amplifying the extent of collateral damage and contributing to significant financial losses. The breadth of investigations focusing on the response of horticultural crops to a single abiotic stress is immense. However, the tolerance mechanisms of horticultural crops to multiple abiotic stresses remain poorly understood. In this review, we described the most prevalent types of abiotic stresses that occur simultaneously and discussed them in in-depth detail regarding the physiological and molecular responses of horticultural crops. In particular, we discussed the transcriptional, posttranscriptional, and metabolic responses of horticultural crops to multiple abiotic stresses. Strategies to breed multi-stress-resilient lines have been presented. Our manuscript presents an interesting amount of proposed knowledge that could be valuable in generating resilient genotypes for multiple stressors.

microRNA regulates cytokinin induced parthenocarpy in cucumber (Cucumis sativus L.).

Parthenocarpy is one of the most important agronomic traits for fruit yield in cucumbers. However, the precise gene regulation and the posttranscriptional mechanism are elusive. In the presented study, one parthenocarpic line DDX and non-parthenocarpic line ZK were applied to identify the microRNAs (miRNAs) involved in parthenocarpic fruit formation. The differential expressed miRNAs among parthenocarpic fruit of forchlorfenuron (CPPU) treated ZK (ZK-CPPU), pollinated ZK (ZK-P), non-pollinated DDX (DDX-NP) were compared with the non-parthenocarpic fruits of non-pollinated ZK (ZK-NP). It indicated 98 miRNAs exhibited differential expression were identified. Notably, a significant proportion of these miRNAs were enriched in the signal transduction pathway of plant hormones, as identified by the KEGG pathway analysis. qRT-PCR validation indicated that CsmiR156 family was upregulated in the ZK-NP while downregulated in ZK-CPPU, ZK-P, and DDX-NP at 1 day after anthesis. Meanwhile, the opposite trend was observed for CsmiR164a. In ZK-CPPU, ZK-P, and DDX-NP, CsmiRNA156 genes (CsSPL16 and CsARR9-like) were upregulated while CsmiRNA164a genes (CsNAC6, CsCUC1, and CsNAC100) were downregulated. The GUS and dual luciferase assay validated that CsmiR156a inhibited while CsmiR164a induced their target genes' transcription. This study presents novel insights into the involvement of CsmiR156a and CsmiR164a in the CK-mediated posttranscriptional regulation of cucumber parthenocarpy, which will aid future breeding programs.

The apple MdGA2ox7 modulates the balance between growth and stress tolerance in an anthocyanin-dependent manner.

Apple (Malus domestica Borkh.) is a widely cultivated fruit crop worldwide but often suffers from abiotic stresses such as salt and cold. Gibberellic acid (GA) plays a pivotal in controlling plant development, environmental adaptability, and secondary metabolism. The GA2-oxidase (GA2ox) is responsible for the deactivation of bioactive GA. In this study, seventeen GA2-oxidase genes were identified in the apple genome, and these members could be clustered into four clades based on phylogenetic relationships and conserved domain structures. MdGA2ox7 exhibited robust expression across various tissues, responded to cold and salt treatments, and was triggered in apple fruit peels via light-induced anthocyanin accumulation. Subcellular localization prediction and experiments confirmed that MdGA2ox7 was located in the cytoplasm. Overexpression of MdGA2ox7 in Arabidopsis caused a lower level of active GA and led to GA-deficient phenotypes, such as dwarfism and delayed flowering. MdGA2ox7 alleviated cold and salt stress damage in both Arabidopsis and apple in concert with melatonin (MT). Additionally, MdGA2ox7 enhanced anthocyanin biosynthesis in apple calli and activated genes involved in anthocyanin synthesis. These findings provide new insights into the functions of apple GA2ox in regulating development, stress tolerance, and secondary metabolism.

RNA-seq-based comparative transcriptome analysis reveals the role of CsPrx73 in waterlogging-triggered adventitious root formation in cucumber.

Abiotic stressors like waterlogging are detrimental to cucumber development and growth. However, comprehension of the highly complex molecular mechanism underlying waterlogging can provide an opportunity to enhance cucumber tolerance under waterlogging stress. We examined the hypocotyl and stage-specific transcriptomes of the waterlogging-tolerant YZ026A and the waterlogging-sensitive YZ106A, which had different adventitious rooting ability under waterlogging. YZ026A performed better under waterlogging stress by altering its antioxidative machinery and demonstrated a greater superoxide ion (O 2-) scavenging ability. KEGG pathway enrichment analysis showed that a high number of differentially expressed genes (DEGs) were enriched in phenylpropanoid biosynthesis. By pairwise comparison and weighted gene co-expression network analysis analysis, 2616 DEGs were obtained which were categorized into 11 gene co-expression modules. Amongst the 11 modules, black was identified as the common module and yielded a novel key regulatory gene, CsPrx73. Transgenic cucumber plants overexpressing CsPrx73 enhance adventitious root (AR) formation under waterlogging conditions and increase reactive oxygen species (ROS) scavenging. Silencing of CsPrx73 expression by virus-induced gene silencing adversely affects AR formation under the waterlogging condition. Our results also indicated that CsERF7-3, a waterlogging-responsive ERF transcription factor, can directly bind to the ATCTA-box motif in the CsPrx73 promoter to initiate its expression. Overexpression of CsERF7-3 enhanced CsPrx73 expression and AR formation. On the contrary, CsERF7-3-silenced plants decreased CsPrx73 expression and rooting ability. In conclusion , our study demonstrates a novel CsERF7-3-CsPrx73 module that allows cucumbers to adapt more efficiently to waterlogging stress by promoting AR production and ROS scavenging.

KNOTTED1-like homeobox (KNOX) transcription factors - Hubs in a plethora of networks: A review.

KNOX (KNOTTED1-like HOMEOBOX) belongs to a class of important homeobox genes, which encode the homeodomain proteins binding to the specific element of target genes, and widely participate in plant development. Advancements in genetics and molecular biology research generate a large amount of information about KNOX genes in model and non-model plants, and their functions in different developmental backgrounds are gradually becoming clear. In this review, we summarize the known and presumed functions of the KNOX gene in plants, focusing on horticultural plants and crops. The classification and structural characteristics, expression characteristics and regulation, interacting protein factors, functions, and mechanisms of KNOX genes are systematically described. Further, the current research gaps and perspectives were discussed. These comprehensive data can provide a reference for the directional improvement of agronomic traits through KNOX gene regulation.

miRNAs for crop improvement.

Climate change significantly impacts crop production by inducing several abiotic and biotic stresses. The increasing world population, and their food and industrial demands require focused efforts to improve crop plants to ensure sustainable food production. Among various modern biotechnological tools, microRNAs (miRNAs) are one of the fascinating tools available for crop improvement. miRNAs belong to a class of small non-coding RNAs playing crucial roles in numerous biological processes. miRNAs regulate gene expression by post-transcriptional target mRNA degradation or by translation repression. Plant miRNAs have essential roles in plant development and various biotic and abiotic stress tolerance. In this review, we provide propelling evidence from previous studies conducted around miRNAs and provide a one-stop review of progress made for breeding stress-smart future crop plants. Specifically, we provide a summary of reported miRNAs and their target genes for improvement of plant growth and development, and abiotic and biotic stress tolerance. We also highlight miRNA-mediated engineering for crop improvement and sequence-based technologies available for the identification of miRNAs associated with stress tolerance and plant developmental events.

MdIPT1, an adenylate isopentenyltransferase coding gene from Malus domestica, is involved in branching and flowering regulation.

Flowering and shoot branching are significant agricultural traits for apple tree breeding. Cytokinin metabolism and signaling pathways play a crucial role in plant development. However, little is known about cytokinin biosynthetic molecular mechanism and function involved in apple flowering and branching. In this study, an adenylate isopentenyl transferase coding gene MdIPT1, homologous to AtIPT3/AtIPT5 in Arabidopsis thaliana, was identified. MdIPT1 was highly expressed in apple floral and axillary buds and was dramatically up-regulated during floral induction and axillary bud outgrowth. The promoter of MdIPT1 showed high activity in multiple tissues and responded to different hormone treatments. The MdIPT1-overexpressing Arabidopsis showed a multi-branching and early-flowering phenotype, with elevated endogenous cytokinin levels and altered expression of genes related to branching and flower formation. Overexpression of MdIPT1 confers the growth vigor of transgenic apple callus on a CKs-deficient medium. Our findings suggest that MdIPT1 is a positive regulator involved in branching and flowering. The data presented herein provide extensive research results on MdIPT1 and will contribute to molecular breeding for new apple varieties.

The BELL1-like homeobox gene MdBLH14 from apple controls flowering and plant height via repression of MdGA20ox3.

Apple growth and yield are largely dependent on plant height and flowering characteristics. The BELL1-like homeobox (BLH) transcription factors regulate extensive plant biological processes. However, the BLH-mediated regulation of plant height and flowering in apple remains elusive. In the current study, 19 members of the MdBLH family were identified in the apple genome. Segmental duplication and purifying selection are the main reasons for the evolution of the MdBLH genes. A BLH1-like gene, MdBLH14, was isolated and functionally characterized. The MdBLH14 was preferentially expressed in flower buds, and downregulated during the floral induction period. The subcellular localization in tobacco leaves indicated that MdBLH14 is a nuclear protein. Overexpression of MdBLH14 in Arabidopsis led to a significant dwarfing and late-flowering phenotype by hindering active GA accumulation. Additionally, MdKNOX19, another member of the TALE superfamily, physically interacts with MdBLH14 and synergistically inhibits the expression of MdGA20ox3. This is the first report on the function of the MdBLH14 from apple, and its mechanism involving plant flower induction and growth. The data presented here provide a theoretical basis for genetically breeding new apple varieties.

Developing drought-smart, ready-to-grow future crops.

Breeding crop plants with increased yield potential and improved tolerance to stressful environments is critical for global food security. Drought stress (DS) adversely affects agricultural productivity worldwide and is expected to rise in the coming years. Therefore, it is vital to understand the physiological, biochemical, molecular, and ecological mechanisms associated with DS. This review examines recent advances in plant responses to DS to expand our understanding of DS-associated mechanisms. Suboptimal water sources adversely affect crop growth and yields through physical impairments, physiological disturbances, biochemical modifications, and molecular adjustments. To control the devastating effect of DS in crop plants, it is important to understand its consequences, mechanisms, and the agronomic and genetic basis of DS for sustainable production. In addition to plant responses, we highlight several mitigation options such as omics approaches, transgenics breeding, genome editing, and biochemical to mechanical methods (foliar treatments, seed priming, and conventional agronomic practices). Further, we have also presented the scope of conventional and speed breeding platforms in helping to develop the drought-smart future crops. In short, we recommend incorporating several approaches, such as multi-omics, genome editing, speed breeding, and traditional mechanical strategies, to develop drought-smart cultivars to achieve the 'zero hunger' goal.

Smart reprograming of plants against salinity stress using modern biotechnological tools.

Climate change gives rise to numerous environmental stresses, including soil salinity. Salinity/salt stress is the second biggest abiotic factor affecting agricultural productivity worldwide by damaging numerous physiological, biochemical, and molecular processes. In particular, salinity affects plant growth, development, and productivity. Salinity responses include modulation of ion homeostasis, antioxidant defense system induction, and biosynthesis of numerous phytohormones and osmoprotectants to protect plants from osmotic stress by decreasing ion toxicity and augmented reactive oxygen species scavenging. As most crop plants are sensitive to salinity, improving salt tolerance is crucial in sustaining global agricultural productivity. In response to salinity, plants trigger stress-related genes, proteins, and the accumulation of metabolites to cope with the adverse consequence of salinity. Therefore, this review presents an overview of salinity stress in crop plants. We highlight advances in modern biotechnological tools, such as omics (genomics, transcriptomics, proteomics, and metabolomics) approaches and different genome editing tools (ZFN, TALEN, and CRISPR/Cas system) for improving salinity tolerance in plants and accomplish the goal of "zero hunger," a worldwide sustainable development goal proposed by the FAO.

Exploring the Agrobacterium-mediated transformation with CRISPR/Cas9 in cucumber (Cucumis sativus L.).

BACKGROUNDS: The narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is  characteristic of  simple design, low cost and  high efficiency, which has opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber. METHODS AND RESULTS: In this study, a Histochemical ß-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1 day, Agrobacterium bacterial solution containing AS, and 20 min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines. CONCLUSION: In this study, 2.4‰ of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to further optimize and improve the efficiency of the genetic transformation of cucumber.

Hormonal interactions underlying parthenocarpic fruit formation in horticultural crops.

In some horticultural crops, such as Cucurbitaceae, Solanaceae, and Rosaceae species, fruit set and development can occur without the fertilization of ovules, a process known as parthenocarpy. Parthenocarpy is an important agricultural trait that can not only mitigate fruit yield losses caused by environmental stresses but can also induce the development of seedless fruit, which is a desirable trait for consumers. In the present review, the induction of parthenocarpic fruit by the application of hormones such as auxins (2,4 dichlorophenoxyacetic acid; naphthaleneacetic acid), cytokinins (forchlorfenuron; 6-benzylaminopurine), gibberellic acids, and brassinosteroids is first presented. Then, the molecular mechanisms of parthenocarpic fruit formation, mainly related to plant hormones, are presented. Auxins, gibberellic acids, and cytokinins are categorized as primary players in initiating fruit set. Other hormones, such as ethylene, brassinosteroids, and melatonin, also participate in parthenocarpic fruit formation. Additionally, synergistic and antagonistic crosstalk between these hormones is crucial for deciding the fate of fruit set. Finally, we highlight knowledge gaps and suggest future directions of research on parthenocarpic fruit formation in horticultural crops.

HD-ZIP Gene Family: Potential Roles in Improving Plant Growth and Regulating Stress-Responsive Mechanisms in Plants.

Exploring the molecular foundation of the gene-regulatory systems underlying agronomic parameters or/and plant responses to both abiotic and biotic stresses is crucial for crop improvement. Thus, transcription factors, which alone or in combination directly regulated the targeted gene expression levels, are appropriate players for enlightening agronomic parameters through genetic engineering. In this regard, homeodomain leucine zipper (HD-ZIP) genes family concerned with enlightening plant growth and tolerance to environmental stresses are considered key players for crop improvement. This gene family containing HD and LZ domain belongs to the homeobox superfamily. It is further classified into four subfamilies, namely HD-ZIP I, HD-ZIP II, HD-ZIP III, and HD-ZIP IV. The first HD domain-containing gene was discovered in maize cells almost three decades ago. Since then, with advanced technologies, these genes were functionally characterized for their distinct roles in overall plant growth and development under adverse environmental conditions. This review summarized the different functions of HD-ZIP genes in plant growth and physiological-related activities from germination to fruit development. Additionally, the HD-ZIP genes also respond to various abiotic and biotic environmental stimuli by regulating defense response of plants. This review, therefore, highlighted the various significant aspects of this important gene family based on the recent findings. The practical application of HD-ZIP biomolecules in developing bioengineered plants will not only mitigate the negative effects of environmental stresses but also increase the overall production of crop plants.

The TAZ domain-containing proteins play important role in the heavy metals stress biology in plants.

TAZ (transcriptional coactivator with PDZ-binding) zinc finger domains, also known as transcription adaptor putative zinc finger domains, that control diverse function in plant growth and development. Here, in the present study, we evaluated the role of the TAZ domain-containing gene in response to various heavy metals. Initially, we found a total of 3, 7, 8, 9, 9, 9, 7, 14, 6, 10, and 6 proteins containing TAZ domain in stiff brome, millet, sorghum, potato, pepper, maize, rice, apple, peach, pear, and tomato genome that could trigger the plant resistance against various heavy metals, respectively. Various in-silico approaches were applied such as duplication, phylogenetic analysis, and gene structure, to understand the basic features of the TAZ domain-containing genes in plants. Gene expression analyses were also performed under heavy metals (Cr, Zn, Ni, Cd, Co, Fe, Mn, and Pb). The results of quantitative real-time PCR analysis indicated that the TAZ gene family members were differentially expressed under different heavy metals. We further characterized the functions of the TAZ domain-containing gene under the heavy metal stresses by overexpressing the OsTAZ4 gene in Arabidopsis. The TAZ genes could promote plant resistance against various heavy metals by interacting with OsMYB34 and OsFHA9 transcription factors. The results will contribute to elucidate the relationship of TAZ proteins with heavy metals stresses and also ascertain the biological function in plant growth and development.

Correction to: CRISPR-Cas9 mediated targeted disruption of FAD2-2 microsomal omega-6 desaturase in soybean (Glycine max.L).

An amendment to this paper has been published and can be accessed via the original article.