RNAi-mediated downregulation of AcCENH3 can induce in vivo haploids in onion (Allium cepa L.).

Haploid induction (HI) holds great promise in expediting the breeding process in onion, a biennial cross-pollinated crop. We used the CENH3-based genome elimination technique in producing a HI line in onion. Here, we downregulated AcCENH3 using the RNAi approach without complementation in five independent lines. Out of five events, only three could produce seeds upon selfing. The progenies showed poor seed set and segregation distortion, and we were unable to recover homozygous knockdown lines. The knockdown lines showed a decrease in accumulation of AcCENH3 transcript and protein in leaf tissue. The decrease in protein content in transgenic plants was correlated with poor seed set. When the heterozygous knockdown lines were crossed with wild-type plants, progenies showed HI by genome elimination of the parental chromosomes from AcCENH3 knockdown lines. The HI efficiency observed was between 0 and 4.63% in the three events, and it was the highest (4.63%) when E1 line was crossed with wildtype. Given the importance of doubled haploids in breeding programmes, the findings from our study are poised to significantly impact onion breeding.

A glossy mutant in onion (Allium cepa L.) shows decreased expression of wax biosynthesis genes.

Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an epicuticular wax layer was identified in the γ-irradiated M2 mutant population of the onion cultivar Bhima Super. The inheritance of the mutant's glossy phenotype was determined to be recessive and single locus. Scanning electron microscopy analysis showed poor accumulation of wax crystals in the glossy mutant, concentrated near the stomata. The plant height, number of leaves per plant, and stomatal parameters of the mutant were similar to the wild-type. RNA-seq was used to comprehend the expression variations of waxy cuticle-related genes in the glossy mutant and its wild-type waxy cultivars. Differential gene expression analysis of the RNA-seq data revealed that the genes involved in wax biosynthesis, such as AcCER1, AcCER26, AcMAH1, and AcWSD1, were downregulated by 2.72, 1.74, 2.59 and 2.12-fold, respectively, in the glossy mutant respectively. The expression patterns of these four unigenes were validated using semi-quantitative RT-PCR. The glossy mutant displayed a substantial 3.5-fold reduction in cuticular wax load compared to the wild-type due to the significant downregulation of these wax biosynthesis genes. These findings represent early advancements in understanding the molecular mechanisms of wax biosynthesis in onions. Furthermore, they provide a foundation for utilizing the glossy mutant trait in breeding programmes to enhance stress and pest resilience.

CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion (Allium cepa L.).

Introduction: Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) is a precise genome editing tool used to introduce genetic modifications in a wide range of crop species. Thus far, there is no report of CRISPR/Cas9-mediated genome editing in onions (Allium cepa L.). Methods: In the present study, we targeted two exons of the gene coding for Phytoene desaturase (AcPDS) in onion cv. Bhima Super. The sgRNA-carrying constructs were co-cultivated with 8-week-old embryogenic calli using an Agrobacterium-mediated transformation protocol and incubated on the media without hygromycin B selection. Results and discussion: Out of the total 617 co-cultivated calli, 21 (3.4%) regenerated shoots exhibited three distinct phenotypes: albino, chimeric, and pale green; in comparison to the wild-type non-transformed regenerated shoots. Total chlorophyll content was drastically reduced in albino shoots and significantly decreased in chimeric shoots. Out of the six Cas9 gene PCR-confirmed regenerated shoots, two exhibited the albino phenotype due to insertions/deletions (InDels) and substitution-based mutations in and around the AcPDS target sites. Deep amplicon sequencing revealed a significantly variable InDel frequency between two sgRNAs, ranging from 1.2% to 63.4%, along with a 53.4% substitution frequency. The mutation of the AcPDS gene generated a visually detectable albino phenotype, thus confirming the successful editing of the AcPDS gene. This is the first time a CRISPR/Cas9-mediated genome editing protocol has been successfully established in onion, with the AcPDS gene serving as an example. This study will provide the necessary momentum for researchers to further basic and applied research on onions.

Identification, cloning and characterization of AcMSH1 from Onion (Allium cepa L.).

BACKGROUND: MSH1 (MutS homolog1) is a nuclear-encoded protein that plays a crucial role in maintaining low mutation rates and stability of the organellar genome. While plastid MSH1 maintains nuclear epigenome plasticity and affects plant development patterns, mitochondrial MSH1 suppresses illegitimate recombination within the mitochondrial genome, affects mitochondrial genome substoichiometric shifting activity and induces cytoplasmic male sterility (CMS) in crops. However, a detailed functional investigation of onion MSH1 has yet to be achieved. MATERIALS AND RESULTS: The homology analysis of onion genome database identified a single copy of the AcMSH1 gene in the onion cv. Bhima Super. In silico analysis of AcMSH1 protein sequence revealed the presence of 6 conserved functional domains including a unique MSH1-specific GIY-YIG endonuclease domain at the C-terminal end. At N-terminal end, it has signal peptide sequences targeting chloroplast and mitochondria. The concentration of AcMSH1 was found to be highest in isolated mitochondria, followed by chloroplasts, and negligible in the cytoplasmic fraction; which proved its localization to the mitochondria and chloroplasts. Quantitative expression analysis revealed that AcMSH1 protein levels were highest in leaves, followed by flower buds, root tips, flowers, and umbels, with the lowest amount found in callus tissue. CONCLUSION: Onion genome has single copy of MSH1, with characteristic GIY-YIG endonuclease domain. AcMSH1 targeted towards both chloroplasts and mitochondria. The identification and characterisation of AcMSH1 may provide valuable insights into the development of CMS lines in onion.

The role of LTR retrotransposons in plant genetic engineering: how to control their transposition in the genome.

KEY MESSAGE: We briefly discuss that the similarity of LTR retrotransposons to retroviruses is a great opportunity for the development of a genetic engineering tool that exploits intragenic elements in the plant genome for plant genetic improvement. Long terminal repeat (LTR) retrotransposons are very similar to retroviruses but do not have the property of being infectious. While spreading between its host cells, a retrovirus inserts a DNA copy of its genome into the cells. The ability of retroviruses to cause infection with genome integration allows genes to be delivered to cells and tissues. Retrovirus vectors are, however, only specific to animals and insects, and, thus, are not relevant to plant genetic engineering. However, the similarity of LTR retrotransposons to retroviruses is an opportunity to explore the former as a tool for genetic engineering. Although recent long-read sequencing technologies have advanced the knowledge about transposable elements (TEs), the integration of TEs is still unable either to control them or to direct them to specific genomic locations. The use of existing intragenic elements to achieve the desired genome composition is better than using artificial constructs like vectors, but it is not yet clear how to control the process. Moreover, most LTR retrotransposons are inactive and unable to produce complete proteins. They are also highly mutable. In addition, it is impossible to find a full active copy of a LTR retrotransposon out of thousands of its own copies. Theoretically, if these elements were directly controlled and turned on or off using certain epigenetic mechanisms (inducing by stress or infection), LTR retrotransposons could be a great opportunity to develop a genetic engineering tool using intragenic elements in the plant genome. In this review, the recent developments in uncovering the nature of LTR retrotransposons and the possibility of using these intragenic elements as a tool for plant genetic engineering are briefly discussed.

SHORT-ROOT stabilizes PHOSPHATE1 to regulate phosphate allocation in Arabidopsis.

The coordinated distribution of inorganic phosphate (Pi) between roots and shoots is an important process that plants use to maintain Pi homeostasis. SHORT-ROOT (SHR) is well characterized for its function in root radial patterning. Here we demonstrate a role of SHR in controlling Pi allocation from root to shoot by regulating PHOSPHATE1 in the root differentiation zone. We recovered a weak mutant allele of SHR in Arabidopsis that accumulates much less Pi in the shoot and shows a constitutive Pi starvation response under Pi-sufficient conditions. In addition, Pi starvation suppresses SHR protein accumulation and releases its inhibition on the HD-ZIP III transcription factor PHB. PHB accumulates and directly binds the promoter of PHOSPHATE2 to upregulate its transcription, resulting in PHOSPHATE1 degradation in the xylem-pole pericycle cells. Our findings reveal a previously unrecognized mechanism of how plants regulate Pi translocation from roots to shoots.

The H3K9me2-binding protein AGDP3 limits DNA methylation and transcriptional gene silencing in Arabidopsis.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

The chromatin remodeler BRAHMA recruits HISTONE DEACETYLASE6 to regulate root growth inhibition in response to phosphate starvation in Arabidopsis.

Plasticity in root system architecture (RSA) allows plants to adapt to changing nutritional status in the soil. Phosphorus availability is a major determinant of crop yield, and RSA remodeling is critical to increasing the efficiency of phosphorus acquisition. Although substantial progress has been made in understanding the signaling mechanism driving phosphate starvation responses in plants, whether and how epigenetic regulatory mechanisms contribute is poorly understood. Here, we report that the Switch defective/sucrose non-fermentable (SWI/SNF) ATPase BRAHMA (BRM) is involved in the local response to phosphate (Pi) starvation. The loss of BRM function induces iron (Fe) accumulation through increased LOW PHOSPHATE ROOT1 (LPR1) and LPR2 expression, reducing primary root length under Pi deficiency. We also demonstrate that BRM recruits the histone deacetylase (HDA) complex HDA6-HDC1 to facilitate histone H3 deacetylation at LPR loci, thereby negatively regulating local Pi deficiency responses. BRM is degraded under Pi deficiency conditions through the 26 S proteasome pathway, leading to increased histone H3 acetylation at the LPR loci. Collectively, our data suggest that the chromatin remodeler BRM, in concert with HDA6, negatively regulates Fe-dependent local Pi starvation responses by transcriptionally repressing the RSA-related genes LPR1 and LPR2 in Arabidopsis thaliana.

An Agrobacterium-mediated transient expression method contributes to functional analysis of a transcription factor and potential application of gene editing in Chenopodium quinoa.

KEY MESSAGE: An efficient Agrobacterium-mediated transient expression method was developed, which contributed to the functional characterization of the transcription factor CqPHR1, and demonstrates the potential application of gene editing in quinoa. Chenopodium quinoa is a crop expected to ensure global food security in future due to its high resistance to multiple abiotic stresses and nutritional value. We cloned one of the paralogous genes of the Arabidopsis homolog PHR1 (PHOSPHATE STARVATION RESPONSE 1) in quinoa-inbred lines by reverse genetic approach. Overexpression of CqPHR1 driven by the constitutive CaMV 35S promoter in Arabidopsis phr1 mutant can complement its phenotypes, including the induction of phosphate starvation-induced (PSI) genes and anthocyanin accumulation in leaves. By Agrobacterium-mediated gene transient expression, we found that CqPHR1 localized in the nucleus of quinoa cells, and overexpression of CqPHR1 in quinoa cells promoted PSI genes expression, which further revealed the function of CqPHR1 as a transcription factor. We have also shown that the transient expression system can be used to express Cas9 protein in various quinoa-inbred lines and perform effective gene editing in quinoa tissue. The method developed in this study will be useful for verifying the effectiveness of gene-editing systems in quinoa cells and has potential application in the generation of gene-edited quinoa with heritable traits.

High transcriptome plasticity drives phosphate starvation responses in tomato.

Tomato is an important vegetable crop and fluctuating available soil phosphate (Pi) level elicits several morpho-physiological responses driven by underlying molecular responses. Therefore, understanding these molecular responses at the gene and isoform levels has become critical in the quest for developing crops with improved Pi use efficiency. A quantitative time-series RNA-seq analysis was performed to decipher the global transcriptomic changes that accompany Pi starvation in tomato. Apart from changes in the expression levels of genes, there were also alterations in the expression of alternatively-spliced transcripts. Physiological responses such as anthocyanin accumulation, reactive oxygen species generation and cell death are obvious 7 days after Pi deprivation accompanied with the maximum amount of transcriptional change in the genome making it an important stage for in-depth study while studying Pi stress responses (PSR). Our study demonstrates that transcriptomic changes under Pi deficiency are dynamic and complex in tomato. Overall, our study dwells on the dynamism of the transcriptome in eliciting a response to adapt to low Pi stress and lays it bare. Findings from this study will prove to be an invaluable resource for researchers using tomato as a model for understanding nutrient deficiency.

Plant phosphate nutrition: sensing the stress.

Phosphorus (P) is obtained by plants as phosphate (Pi) from the soil and low Pi levels affects plant growth and development. Adaptation to low Pi condition entails sensing internal and external Pi levels and translating those signals to molecular and morphophysiological changes in the plant. In this review, we present findings related to local and systemin Pi sensing with focus the molecular mechanisms behind root system architectural changes and the impact of hormones and epigenetic mechanisms affecting those changes. We also present some of the recent advances in the Pi sensing and signaling mechanisms focusing on inositol pyrophosphate InsP8 and its interaction with SPX domain proteins to regulate the activity of the central regulator of the Pi starvation response, PHR.

A simple flow cytometry-based assay to study global methylation levels in onion, a non-model species.

DNA methylation is an important epigenetic mark and global methylation dynamics regulate plant developmental processes. Even though genome sequencing technologies have made DNA methylation studies easier, it is difficult in non-model species where genome information is not available. Therefore in this study, we developed a simple assay for analysing global methylation levels in plants by washless immunolabelling of unfixed nuclei using flow cytometry. Onion leaf tissue was used as a model system, and mean fluorescence intensity due to anti-5- methyl cytosine (5-mC) antibodies were used as a measure of global methylation levels. Among three nuclear isolation buffers evaluated, the highest nuclear yield with the low background was obtained with LB01. To maintain a balance between high DNA fluorescence value and low coefficient of variation of DNA peaks, 45 min of hydrolysis with 0.2 N hydrochloric acid was used for chromatin denaturation resulting in six-fold increase in 5-mC fluorescence compared to control. This method was used successfully to detect 5-Azacytidine induced DNA hypomethylation in onion leaf tissues. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01047-6.

Distinct morpho-physiological and biochemical features of arid and hyper-arid ecotypes of Ziziphus nummularia under drought suggest its higher tolerance compared with semi-arid ecotype.

Tree species in the arid and semi-arid regions use various strategies to combat drought stress. Ziziphus nummularia (Burm. f.) Wight et Arn., native to the Thar Desert in India, is highly drought-tolerant. To identify the most drought-tolerant ecotype of Z. nummularia, one ecotype each from semi-arid (Godhra, annual rainfall >750 mm), arid (Bikaner, 250-350 mm) and hyper-arid (Jaisalmer, <150 mm) regions was selected along with two other Ziziphus species, Ziziphus mauritiana Lamk. and Ziziphus rotundifolia Lamk., and screened for parameters contributing to drought tolerance. Among these, Z. nummularia (Jaisalmer) (CIAHZN-J) was the most drought - tolerant. The tolerance nature of CIAHZN-J was associated with increased membrane stability, root length and number, length of hairs and thorns, root dry/fresh weight ratio, seed germination (at -0.5 MPa), proline content (31-fold), catalase and sugar content (two- to three-fold). Apart from these characteristics, it also exhibited the longest duration to reach highest cumulative drought stress rating, maintained higher relative water content for a longer period of time with reduced leaf size, leaf rolling and falling of older leaves, and displayed sustained shoot growth during drought stress. To determine drought tolerance in Ziziphus, we developed a morphological symptom-based screening technique in this study. Additionally, transcriptome profiling of CIAHZN-J in response to drought revealed the up-regulation of genes involved in sugar metabolism and transport, abscisic acid biosynthesis, osmoregulation, reactive oxygen species homeostasis and maintaining water potential. Expression profiles and semi-quantitative reverse transcription PCR results further correlated with the physiological and biochemical mechanisms. In conclusion, CIAHZN-J is an excellent genetic stock for the identification of drought-responsive genes and can also be deployed in crop improvement programs for drought tolerance.

Bioinformatics Analysis Guides to LTR Retrotransposon-Derived Extrachromosomal Linear DNAs Identified by ALE-seq.

ALE-seq is a method devised to identify pre-integration intermediates of LTR retrotransposons called extrachromosomal linear DNA, which can be used to predict retrotransposition activity. We describe here a bioinformatic methodology to process reads obtained from the ALE-seq protocol for the effective annotation of novel and active retroelements.

Bamboo Transposon Research: Current Status and Perspectives.

Bamboo, a fast-growing non-timber forest plant with many uses, is a valuable species for green development. However, bamboo flowering is very infrequent, extending, in general, for up to 120 years. Ecologically, bamboo species are generally better adapted to various environments than other grasses. Therefore, the species deserves a special status in what could be called Ecological Bioeconomy. An understanding of the genetic processes of bamboo can help us sustainably develop and manage bamboo forests. Transposable elements (TEs), jumping genes or transposons, are major genetic elements in plant genomes. The rapid development of the bamboo reference genome, at the chromosome level, reveals that TEs occupy over 63.24% of the genome. This is higher than found in rice, Brachypodium, and sorghum. The bamboo genome contains diverse families of TEs, which play a significant role in bamboo's biological processes including growth and development. TEs provide important clues for understanding the evolution of the bamboo genome. In this chapter, we briefly describe the current status of research on TEs in the bamboo genome, their regulation, and transposition mechanisms. Perspectives for future research are also provided.

Recent advancement of NGS technologies to detect active transposable elements in plants.

BACKGROUND: Unlike peoples' belief that transposable elements (TEs) are "junk DNAs" or "genomic parasites", TEs are essential genomic elements that bring about genetic diversity and enable evolution of a species. In fact, transposons are major constituent of chromosome in crop genomes, particularly in major cereal crops, the primary type of which is long terminal repeat (LTR) retrotransposon. Since TE mobilization can be controlled by specific environmental stimulation and as the result can generate novel genetic variations, it has been suggested that controlled mobilization of TEs can be a plausible method for crop breeding. To achieve this goal, series of sequencing techniques have been recently established to identify TEs that are active in mobility. These methods target and detect extrachromosomal DNAs (ecDNAs), which are final products of integration. The newly identified TEs by these methods exhibit strong transpositional activity which can generate novel genetic diversity and provide useful breeding resources. CONCLUSIONS: In this mini review, we summarize and introduce ALE-seq, mobilome-seq, and VLP DNA-seq techniques employed to detect active TEs in plants.

Multi-omics analysis of cellular pathways involved in different rapid growth stages of moso bamboo.

Moso bamboo (Phyllostachys edulis (Carriere) J. Houzeau) is a rapidly growing grass of industrial and ecological importance. However, the molecular mechanisms of its remarkable growth are not well understood. In this study, we investigated the early-stage growth of moso bamboo shoots and defined three different growth stages based on histological and biochemical analyses, namely, starting of cell division (SD), rapid division (RD) and rapid elongation (RE). Further analyses on potentially relevant cellular pathways in these growth stages using multi-omics approaches such as transcriptomics and proteomics revealed the involvement of multiple cellular pathways, including DNA replication, repair and ribosome biogenesis. A total of 8045 differentially expressed genes (DEGs) and 1053 differentially expressed proteins (DEPs) were identified in our analyses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of detected DEGs identified several key biological pathways such as phytohormone metabolism, signal transduction, cell wall development and carbohydrate metabolism. The comparative analysis of proteins displayed that a total of 213 DEPs corresponded with DEGs and 3 significant expression profiles that could be promoting the fast growth of bamboo internodes. Moreover, protein-protein interaction network prediction analysis is suggestive of the involvement of five major proteins of signal transduction, DNA synthesis and RNA transcription, and may act as key elements responsible for the rapid shoot growth. Our work exploits multi-omics and bioinformatic approaches to unfurl the complexity of molecular networks involved in the rapid growth of moso bamboo and opens up questions related to the interactions between the functions played by individual molecular pathway.

A RAF-SnRK2 kinase cascade mediates early osmotic stress signaling in higher plants.

Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.

Inositol Pyrophosphate InsP8 Acts as an Intracellular Phosphate Signal in Arabidopsis.

The maintenance of cellular phosphate (Pi) homeostasis is of great importance in living organisms. The SPX domain-containing protein 1 (SPX1) proteins from both Arabidopsis and rice have been proposed to act as sensors of Pi status. The molecular signal indicating the cellular Pi status and regulating Pi homeostasis in plants, however, remains to be identified, as Pi itself does not bind to the SPX domain. Here, we report the identification of the inositol pyrophosphate InsP8 as a signaling molecule that regulates Pi homeostasis in Arabidopsis. Polyacrylamide gel electrophoresis profiling of InsPs revealed that InsP8 level positively correlates with cellular Pi concentration. We demonstrated that the homologs of diphosphoinositol pentakisphosphate kinase (PPIP5K), VIH1 and VIH2, function redundantly to synthesize InsP8, and that the vih1 vih2 double mutant overaccumulates Pi. SPX1 directly interacts with PHR1, the central regulator of Pi starvation responses, to inhibit its function under Pi-replete conditions. However, this interaction is compromised in the vih1 vih2 double mutant, resulting in the constitutive induction of Pi starvation-induced genes, indicating that plant cells cannot sense cellular Pi status without InsP8. Furthermore, we showed that InsP8 could directly bind to the SPX domain of SPX1 and is essential for the interaction between SPX1 and PHR1. Collectively, our study suggests that InsP8 is the intracellular Pi signaling molecule serving as the ligand of SPX1 for controlling Pi homeostasis in plants.