Wild-type Seedlings Research Articles

A visible seedling-stage screening system for the Brassica napus hybrid breeding by a novel hypocotyl length-regulated gene BnHL.

Rapeseed (Brassica napus) is a globally significant oilseed crop with strong heterosis performance. Recessive genic male sterility (RGMS) is one of the key approaches for utilizing heterosis in B. napus. However, this method faces the inherent challenge of being time-consuming and labour-intensive for removing fertile plants during seed production. Here, we report a hypocotyl length-regulated gene, BnHL, which is closely linked to a known fertility gene, BnMs2, serving as a seedling morphology marker. This marker could be used to identify fertile plants in the breeding of RGMS lines based on hypocotyl traits. By targeting the BnHL gene, both homozygous and heterozygous edited mutants exhibited significantly longer hypocotyls than the wild type (WT). Furthermore, germination experiments revealed that 7 days after seed germination, the difference in hypocotyl length between the mutant and the WT seedlings reached its maximum, effectively distinguishing fertile plants under both white (W) and red/far-red (R/FR) light. Mutations in BnHL did not result in significant changes in main agronomic traits. Thus, this study provides a comprehensive strategy for screening and identifying a new morphological marker gene for early screening in RGMS hybrid breeding with completely non-transgene during the whole production.

ABI3 regulates ABI1 function to control cell length in primary root elongation zone.

Post-embryonic primary root growth is effectively an interplay of several hormone signalling pathways. Here, we show that the ABA-responsive transcription factor ABI3 controls primary root growth through the regulation of JA signalling molecule JAZ1 along with ABA-responsive factor ABI1. In the absence of ABI3, the primary root elongation zone is shortened with significantly reduced cell length. Expression analyses and ChIP-based assays indicate that ABI3 negatively regulates JAZ1 expression by occupying its upstream regulatory sequence and enriching repressive histone modification mark H3K27 trimethylation, thereby occluding RNAPII occupancy. Previous studies have shown that JAZ1 interacts with ABI1, the protein phosphatase 2C, that works during ABA signalling. Our results indicate that in the absence of ABI3, when JAZ1 expression levels are high, the ABI1 protein shows increased stability, compared to when JAZ1 is absent, or ABI3 is overexpressed. Consequently, in the abi3-6 mutant, due to the higher stability of ABI1, reduced phosphorylation of plasma membrane H+-ATPase (AHA2) occurs. HPTS staining further indicated that abi3-6 root cell apoplasts show reduced protonation, compared to wild-type and ABI3 overexpressing seedlings. Such impeded proton extrusion negatively affects cell length in the primary root elongation zone. ABI3 therefore controls cell elongation in the primary root by affecting the ABI1-dependent protonation of root cell apoplasts. In summary, ABI3 controls the expression of JAZ1 and in turn modulates the function of ABI1 to regulate cell length in the elongation zone during primary root growth.

Cadmium-induced protein AS8: A protein to improve Cd accumulation and transport via Cd uptake in poplar

The pollution of soil with heavy metals (HMs) has become an environmental problem of global concern. Phytoremediation, whereby plants extract HMs from soil, can efficiently and substantially reduce HM pollution in soil in an environmentally friendly manner. Cadmium-induced protein AS8 (CIPAS8) is present in many plants and its expression is induced by HMs. In this study, PeCIPAS8 and SlCIPAS8 were transformed into 84K poplar to study their effects on tolerance to, and translocation of, cadmium (Cd) in woody plants. Localization analyses showed that two CIPAS8 proteins were localized at the plasma membrane when transiently expressed in tobacco leaf epidermal cells. Compared with wild-type 84K poplar seedlings, transgenic poplar lines overexpressing PeCIPAS8 or SlCIPAS8 showed increased Cd contents and decreased Cd tolerance. Transgenic poplar lines overexpressing PeCIPAS8 or SlCIPAS8 accumulated more Cd in the roots, stems, and leaves, but the plant height did not differ significantly, compared with wild-type 84K poplar under Cd stress during the vegetative stage. CIPAS8 increased the Cd influx rate of transgenic poplar roots compared with that of the wild type, and affected the transcription levels of other metal transporters. These findings show that CIPAS8 increases Cd flux into plant tissues and demonstrate moderate Cd sensitivity of the plant. Therefore, CIPAS8 is an influx transporter with the potential to increase the uptake of toxic HMs by woody plants growing in HM-contaminated soils.

Modulation of the Arabidopsis Starch Metabolic Network by the Cytosolic Acetyl-CoA Pathway in the Context of the Diurnal Illumination Cycle

The starch metabolic network was investigated in relation to other metabolic processes by examining a mutant with altered single-gene expression of ATP citrate lyase (ACL), an enzyme responsible for generating cytosolic acetyl-CoA pool from citrate. Previous research has shown that transgenic antisense plants with reduced ACL activity accumulate abnormally enlarged starch granules. In this study, we explored the underlying molecular mechanisms linking cytosolic acetyl-CoA generation and starch metabolism under short-day photoperiods. We performed transcriptome and quantification of starch accumulation in the leaves of wild-type and antisense seedlings with reduced ACL activity. The antisense-ACLA mutant accumulated more starch than the wild type under short-day conditions. Zymogram analyses were conducted to compare the activities of starch-metabolizing enzymes with transcriptomic changes in the seedling. Differential expression between wild-type and antisense-ACLA plants was detected in genes implicated in starch and acetyl-CoA metabolism, and cell wall metabolism. These analyses revealed a strong correlation between the transcript levels of genes responsible for starch synthesis and degradation, reflecting coordinated regulation at the transcriptomic level. Furthermore, our data provide novel insights into the regulatory links between cytosolic acetyl-CoA metabolism and starch metabolic pathways.

Gβγ dimers mediate low K+ stress-inhibited root growth via modulating auxin redistribution in Arabidopsis.

In the investigation of heterotrimeric G protein-mediated signal transduction in planta, their roles in the transmittance of low K+ stimuli remain to be elucidated. Here, we found that the primary root growth of wild-type Arabidopsis was gradually inhibited with the decrease of external K+ concentrations, while the primary root of the mutants for G protein β subunit AGB1 and γ subunits AGG1, AGG2 and AGG3 could still grow under low K+ conditions (LK). Exogenous NAA application attenuated primary root elongation in agb1 and agg1/2/3 but promoted the growth in wild-type seedlings under LK stress. Using ProDR5:GFP, ProPIN1:PIN1-GFP and ProPIN2:PIN2-GFP reporter lines, a diminishment in auxin concentration at the radicle apex and a reduction in PIN1and PIN2 efflux carrier abundance were observed in wild-type roots under LK, a phenomenon not recorded in the agb1 and agg1/2/3. Further proteolytic and transcriptional assessments revealed an enhanced degradation of PIN1 and a suppressed expression of PIN2 in the wild-type background under LK, contrasting with the stability observed in the agb1 and agg1/2/3 mutants. Our results indicate that the G protein β and γ subunits play pivotal roles in suppressing of Arabidopsis root growth under LK by modulating auxin redistribution via alterations in PIN1 degradation and PIN2 biosynthesis.

Decoding the role of OsPRX83 in enhancing osmotic stress tolerance in rice through ABA-dependent pathways and ROS scavenging

ABSTRACT Plant Class III peroxidases have diverse roles in controlling root hair growth, anther development, and abiotic and biotic stress responses. However, their abiotic stress response mechanism in rice remains elusive. Here, we identified a peroxidase precursor gene, OsPRX83, and investigated its role in enhancing osmotic stress tolerance in rice. We used OsPRX83 overexpression and CRISPR-Cas9-generated mutant lines to elucidate OsPRX83‘s function and expression patterns under stress conditions. The expression of OsPRX83 was induced by H2O2, PEG, NaCl, and ABA treatments. Using qRT-PCR, RNA sequencing, and physiological assays, we demonstrated that overexpression of OsPRX83 enhanced the osmotic and oxidative stress tolerance as compared to the wild-type and mutant seedlings, as evident from the higher survival rates, enhanced peroxidase (POD) and ascorbate peroxidase (APX) activities, and increased ABA sensitivity compared with mutants and wild-type plants. Transcriptome analysis further supported the involvement of OsPRX83 in the ROS scavenging, by modulating the expression of OsDREB1B, OsDREB1E, OsDREB1F, OsDREB1G in response to osmotic treatment. In summary, our study suggests that OsPRX83 plays a pivotal role in enhancing stress tolerance in rice through ABA-dependent pathways and ROS scavenging. Therefore, this study elucidates the function of a novel abiotic stress response gene in rice, thereby may contribute to a new genetic engineering resource for engineering drought-resistant rice varieties.

Salicylic acid improves chilling tolerance via CsNPR1-CsICE1 interaction in grafted cucumbers.

Salicylic acid (SA) plays a role in the regulation of grafting-induced cold tolerance. However, the molecular mechanism behind it is still unknown. Here, we established that the phenylalanine ammonia-lyase (PAL) pathway-dependent elevate in SA content in grafted cucumber leaves was not only synthesized in the leaves but also transported from the roots under chilling stress. RNAi-CsPAL with low SA content as rootstock reduced SA accumulation in grafted seedling leaves while decreasing rootstock-induced cold tolerance, as evidenced by higher electrolyte leakage (EL), hydrogen peroxide (H2O2), and superoxide anion (O2 ·-) contents and lower expression of cold-responsive genes (CsICE1, CsDREB1A, CsDREB1B, and CsCOR47), whereas OE-CsPAL with high SA content as rootstock improved the cold tolerance of grafted plants in comparison with the wild type (WT). In addition, CsNPR1 was significantly upregulated in grafted cucumber under chilling stress, with exogenous and endogenous overexpressed SA inducing its transcriptional expression and protein stability, which exhibited higher expression in grafted plants than in self-root plants. While CsNPR1-overexpression (OE-CsNPR1) seedlings as scions were more tolerant to chilling stress than WT seedlings, CsNPR1-suppression (Anti-CsNPR1) seedlings as scions were more vulnerable to chilling stress. Notably, CsNPR1-CsICE1 interactions alleviated ROS accumulation and activated the expression of CsDREB1A, CsDREB1B, CsCOR47, CsCOR15, CsCOR413, and CsKIN1 to enhance SA-mediated chilling tolerance in grafted cucumber. Overall, our findings reveal that SA enhances chilling tolerance in grafted cucumbers via the model of the CsNPR1-CsICE1 transcriptional regulatory cascade.

Ectopic overexpression of ShCBF1 and SlCBF1 in tomato suggests an alternative view of fruit responses to chilling stress postharvest.

Postharvest chilling injury (PCI) is a physiological disorder that often impairs tomato fruit ripening; this reduces fruit quality and shelf-life, and even accelerates spoilage at low temperatures. The CBF gene family confers cold tolerance in Arabidopsis thaliana, and constitutive overexpression of CBF in tomato increases vegetative chilling tolerance, in part by retarding growth, but, whether CBF increases PCI tolerance in fruit is unknown. We hypothesized that CBF1 overexpression (OE) would be induced in the cold and increase resistance to PCI. We induced high levels of CBF1 in fruit undergoing postharvest chilling by cloning it from S. lycopersicum and S. habrochaites, using the stress-inducible RD29A promoter. Harvested fruit were cold-stored (2.5°C) for up to three weeks, then rewarmed at 20°C for three days. Transgene upregulation was triggered during cold storage from 8.6- to 28.6-fold in SlCBF1-OE, and between 3.1- to 8.3-fold in ShCBF1-OE fruit, but developmental abnormalities in the absence of cold induction were visible. Remarkably, transgenic fruit displayed worsening of PCI symptoms, i.e., failure to ripen after rewarming, comparatively higher susceptibility to decay relative to wild-type (WT) fruit, lower total soluble solids, and the accumulation of volatile compounds responsible for off-odors. These symptoms correlated with CBF1 overexpression levels. Transcriptomic analysis revealed that the ripening and biotic and abiotic stress responses were altered in the cold-stored transgenic fruit. Seedlings grown from 'chilled' and 'non-chilled' WT fruit, in addition to 'non-chilled' transgenic fruit were also exposed to 0°C to test their photosynthetic response to chilling injury. Chilled WT seedlings adjusted their photosynthetic rates to reduce oxidative damage; 'non-chilled' WT seedlings did not. Photosynthetic parameters between transgenic seedlings were similar at 0°C, but SlCBF1-OE showed more severe photoinhibition than ShCBF1-OE, mirroring phenotypic observations. These results suggest that 1) CBF1 overexpression accelerated fruit deterioration in response to cold storage, and 2) Chilling acclimation in fructus can increase chilling tolerance in seedling progeny of WT tomato.

Arabidopsis thaliana MYC2 and MYC3 Are Involved in Ethylene-Regulated Hypocotyl Growth as Negative Regulators.

The ethylene-regulated hypocotyl elongation of Arabidopsis thaliana involves many transcription factors. The specific role of MYC transcription factors in ethylene signal transduction is not completely understood. The results here revealed that two MYCs, MYC2 and MYC3, act as negative regulators in ethylene-suppressed hypocotyl elongation. Etiolated seedlings of the loss-of-function mutant of MYC2 or MYC3 were significantly longer than wild-type seedlings. Single- or double-null mutants of MYC2 and MYC3 displayed remarkably enhanced response to ACC(1-aminocyclopropane-1-carboxylate), the ethylene precursor, compared to wild-type seedlings. MYC2 and MYC3 directly bind to the promoter zone of ERF1, strongly suppressing its expression. Additionally, EIN3, a key component in ethylene signaling, interacts with MYC2 or MYC3 and significantly suppresses their binding to ERF1's promoter. MYC2 and MYC3 play crucial roles in the ethylene-regulated expression of functional genes. The results revealed the novel role and functional mechanism of these transcription factors in ethylene signal transduction. The findings provide valuable information for deepening our understanding of their role in regulating plant growth and responding to stress.

Mitochondrial ATP Synthase beta-Subunit Affects Plastid Retrograde Signaling in Arabidopsis.

Plastid retrograde signaling plays a key role in coordinating the expression of plastid genes and photosynthesis-associated nuclear genes (PhANGs). Although plastid retrograde signaling can be substantially compromised by mitochondrial dysfunction, it is not yet clear whether specific mitochondrial factors are required to regulate plastid retrograde signaling. Here, we show that mitochondrial ATP synthase beta-subunit mutants with decreased ATP synthase activity are impaired in plastid retrograde signaling in Arabidopsis thaliana. Transcriptome analysis revealed that the expression levels of PhANGs were significantly higher in the mutants affected in the AT5G08670 gene encoding the mitochondrial ATP synthase beta-subunit, compared to wild-type (WT) seedlings when treated with lincomycin (LIN) or norflurazon (NF). Further studies indicated that the expression of nuclear genes involved in chloroplast and mitochondrial retrograde signaling was affected in the AT5G08670 mutant seedlings treated with LIN. These changes might be linked to the modulation of some transcription factors (TFs), such as LHY (Late Elongated Hypocotyl), PIF (Phytochrome-Interacting Factors), MYB, WRKY, and AP2/ERF (Ethylene Responsive Factors). These findings suggest that the activity of mitochondrial ATP synthase significantly influences plastid retrograde signaling.

RNA N6-adenine methylation dynamics impact Hyaloperonospora arabidopsidis resistance in Arabidopsis.

In plants, epitranscriptomic mark N6-adenine methylation (m6A) is dynamically regulated in response to environmental cues. However, little is known about m6A dynamics under biotic stresses and their role in environmental adaptation. Additionally, current methodologies limit the investigation of m6A dynamics at single-nucleotide resolution on specific RNA molecules. Using Oxford Nanopore Technology direct RNA sequencing and a neural network model, we show transcript-specific dynamics of m6A modification at single-nucleotide resolution during Hyaloperonospora arabidopsidis (Hpa) infection in Arabidopsis (Arabidopsis thaliana). In wild-type seedlings, pathogen infection causes a significant reduction in global m6A ratios, which corresponds with the activation of m6A-modified transcripts. Defect of m6A deposition in the m6A mutant hakai-1 mimics m6A reduction from Hpa infection at ∼70% of sites, resulting in constitutive overexpression of basal defense genes and enhanced resistance against the pathogen. Our results demonstrate that m6A dynamics impact defense response against Hpa, providing a promising target for future crop improvement strategies.

The UDP-glycosyltransferase gene OsUGT706E2 negatively regulates rice tolerance to blast disease and abiotic stresses

Despite the crucial role of UDP-glycosyltransferases (UGTs) in various stress responses in plants, their biological functions in rice (Oryza sativa L.) remain poorly understood. In this study, we introduce a novel gene, OsUGT706E2, which is expressed at significantly higher levels in wild rice compared to cultivated rice. OsUGT706E2 is active in multiple tissues and localizes to both the nucleus and cytoplasm. Its transcription is notably up-regulated in response to cold stress, blast disease, and several hormone treatments. Overexpression of OsUGT706E2 markedly reduces seedling tolerance to blast disease, cold, and osmotic stress, whereas knocking out OsUGT706E2 enhances seedling tolerance to blast disease and osmotic stress. The expression levels of stress-related genes in OsUGT706E2 overexpressing plants were significantly lower compared to wild-type plants following stress treatment. Conversely, OsUGT706E2 knockout plants exhibited markedly higher expression levels of these genes than the control plants after stress treatment. Metabolome analysis further indicated that OsUGT760E2 influences metabolite content in rice. Specifically, OsUGT760E2-overexpressing seedlings had higher amino acid content and lower lipid content compared to wild-type seedlings, while OsUGT760E2-knockout seedlings showed lower amino acid content and higher lipid content than control seedlings. These findings suggest that OsUGT760E2 negatively regulates resistance to blast disease as well as tolerance to cold and osmotic stress in rice. As a result, OsUGT760E2 represents a promising target for enhancing rice stress tolerance through gene editing approaches.

Cell wall integrity modulates HOOKLESS1 and PHYTOCHROME INTERACTING FACTOR4 expression controlling apical hook formation.

Formation of the apical hook in etiolated dicot seedlings results from differential growth in the hypocotyl apex and is tightly controlled by environmental cues and hormones, among which auxin and gibberellins (GAs) play an important role. Cell expansion is tightly regulated by the cell wall, but whether and how feedback from this structure contributes to hook development is still unclear. Here, we show that etiolated seedlings of the Arabidopsis (Arabidopsis thaliana) quasimodo2-1 (qua2) mutant, defective in pectin biosynthesis, display severe defects in apical hook formation and maintenance, accompanied by loss of asymmetric auxin maxima and of differential cell expansion. Moreover, qua2 seedlings show reduced expression of HOOKLESS 1 (HLS1) and PHYTOCHROME INTERACTING FACTOR 4 (PIF4), which are positive regulators of hook formation. Treatment of wild-type seedlings with the cellulose inhibitor isoxaben (isx) also prevents hook development and represses HLS1 and PIF4 expression. Exogenous GAs, loss of DELLA proteins or HLS1 overexpression partially restore hook development in qua2 and isx-treated seedlings. Interestingly, increased agar concentration in the medium restores, both in qua2 and isx-treated seedlings, hook formation, asymmetric auxin maxima and PIF4 and HLS1 expression. Analysis of plants expressing a FRET-based GA sensor indicate that isx reduces accumulation of GAs in the apical hook region in a turgor-dependent manner. Lack of the cell wall integrity sensor THESEUS 1, which modulates turgor loss point, restores hook formation in qua2 and isx-treated seedlings. We propose that turgor-dependent signals link changes in cell wall integrity to the PIF4-HLS1 signalling module to control differential cell elongation during hook formation.

Endoplasmic reticulum protein ALTERED MERISTEM PROGRAM 1 negatively regulates senescence in Arabidopsis.

Plant senescence is a highly regulated developmental program crucial for nutrient reallocation and stress adaptation in response to developmental and environmental cues. Stress-induced and age-dependent natural senescence share both overlapping and distinct molecular responses and regulatory schemes. Previously, we have utilized a carbon-deprivation (C-deprivation) senescence assay using Arabidopsis (Arabidopsis thaliana) seedlings to investigate senescence regulation. Here we conducted a comprehensive time-resolved transcriptomic analysis of Arabidopsis wild type seedlings subjected to C-deprivation treatment at multiple time points, unveiling substantial temporal changes and distinct gene expression patterns. Moreover, we identified ALTERED MERISTEM PROGRAM 1 (AMP1), encoding an endoplasmic reticulum protein, as a potential regulator of senescence based on its expression profile. By characterizing loss-of-function alleles and overexpression lines of AMP1, we confirmed its role as a negative regulator of plant senescence. Genetic analyses further revealed a synergistic interaction between AMP1 and the autophagy pathway in regulating senescence. Additionally, we discovered a functional association between AMP1 and the endosome-localized ABNORMAL SHOOT3 (ABS3)-mediated senescence pathway and positioned key senescence-promoting transcription factors downstream of AMP1. Overall, our findings shed light on the molecular intricacies of transcriptome reprogramming during C-deprivation-induced senescence and the functional interplay among endomembrane compartments in controlling plant senescence.

Immunocompromisation of wheat host by L-BSO and 2,4-DPA induces susceptibility to the fungal pathogen Fusarium oxysporum

Susceptibility is defined as the disruption of host defence systems that promotes infection or limits pathogenicity. Glutathione (GSH) is a major component of defence signalling pathways that maintain redox status and is synthesised by γ-glutamyl cysteine synthetase (γ-ECS). On the other hand, lignin acts as a barrier in the primary cell wall of vascular bundles (VBs) synthesised by phenylalanine ammonia-lyase (PAL) in the intracellular system of plants. In this study, we used two inhibitors, such as L-Buthionine-sulfoximine (BSO), which irreversibly inhibits γ-ECS, and 2,4-dichlorophenoxyacetic acid (DPA), which reduces PAL activity and leads to the induction of oxidative stress in wheat (Triticum aestivum) seedlings after exposure to Fusarium oxysporum. Seedlings treated with 1 mM L-BSO and 2,4-DPA showed high levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), carbonyl (CO) content, and low activity of antioxidative enzymes [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR)] as compared to wild-type (WT) seedlings under F. oxysporum infection. Further, the content of reduced glutathione (RGSH), ascorbate (ASC), and lignin was decreased in BSO and DPA treated seedlings as compared to WT seedlings during Fusarium infection. Moreover, treatment with BSO and DPA significantly inhibited the relative activity of γ-ECS and PAL (P ≤ 0.001) in WT seedlings during Fusarium infection, which led to disintegrated VBs and, finally, cell death. Our results demonstrate that inhibition of γ-ECS and PAL by BSO and DPA, respectively, disrupts the defence mechanisms of wheat seedlings and induces susceptibility to F. oxysporum.

A DUF966 gene family member OsDSR3 positively regulates alkali stress tolerance in rice

Rice growth and production are severely constrained by alkali stress. However, the mechanism underlying the rice tolerance to alkali stress is unclear. OsDSR3, a novel gene from the domains of unknown function 966 (DUF966) family, was identified and characterized for its function in the response of rice to alkali stress. The result of this study clearly showed that alkali stress significantly induced OsDSR3 expression level. Moreover, the expression of OsDSR3 was up-regulated by drought, salt, cold, H2O2 and abscisic acid (ABA), and down-regulated by gibberellic acid (GA3), and 2,4-Dichlorophenoxyacetic acid (2,4-D) treatments. Subcellular localization exhibited that OsDSR3 was detected in the nucleus and membrane. OsDSR3-overexpressing (OsDSR3-OE) plants showed higher tolerance to alkali stress than the wild-type (WT). In contrast, OsDSR3 knockout (OsDSR3-KO) mutants were more vulnerable to alkali stress. The differentially expressed genes (DEGs) among OsDSR3-OE and WT seedlings were mainly enriched in porphyrin and chlorophyll, starch and sucrose, and carotenoid metabolic pathways. Among these DEGs, 26 were identified as potential alkali stress-responsive genes, including several up-regulated genes like OsHAK5, OsGRX23 and OsNIR2. Consistent with the expression profiles of metabolic pathways-related genes, most of the metabolite contents and metabolite synthases activities were improved in OsDSR3-OE lines and decreased in OsDSR3-KO lines compared to WT. This may explain the higher tolerance of OE lines and lower tolerance of KO lines to alkali stress. These findings suggested that OsDSR3 positively regulates rice tolerance to alkali stress, which will help to elucidate the molecular mechanism underlying rice alkali tolerance.

Generation and characterization of single and multigene Arabidopsis thaliana mutants in LSU1-4 (RESPONSE TO LOW SULFUR) genes

In Arabidopsis thaliana, there are four members of the LSU (RESPONSE TO LOW SULFUR) gene family which are tandemly located on chromosomes 3 (LSU1 and LSU3) and 5 (LSU2 and LSU4). The LSU proteins are small, with coiled-coil structures, and they are able to form homo- and heterodimers. LSUs are involved in plant responses to environmental challenges, such as sulfur deficiency, and plant immune responses. Assessment of the role and function of these proteins was challenging due to the absence of deletion mutants. Our work fulfills this gap through the construction of a set of LSU deletion mutants (single, double, triple, and quadruple) by CRISPR/Cas9 technology. The genomic deletion regions in the obtained lines were mapped and the level of expression of each LSUs was assayed in each mutant. All lines were viable and capable of seed production. Their growth and development were compared at several different stages with the wild-type. No significant and consistent differences in seedlings’ growth and plant development were observed in the optimal conditions. In sulfur deficiency, the roots of 12-day-old wild-type seedlings exhibited increased length compared to optimal conditions; however, this difference in root length was not observed in the majority of lsu-KO mutants.

MORF9-dependent specific plastid RNA editing inhibits root growth under sugar starvation in Arabidopsis.

Multiple organellar RNA editing factor (MORF) complex was shown to be highly associated with C-to-U RNA editing of vascular plant editosome. However, mechanisms by which MORF9-dependent plastid RNA editing controls plant development and responses to environmental alteration remain obscure. In this study, we found that loss of MORF9 function impaired PSII efficiency, NDH activity, and carbohydrate production, rapidly promoted nuclear gene expression including sucrose transporter and sugar/energy responsive genes, and attenuated root growth under sugar starvation conditions. Sugar repletion increased MORF9 and MORF2 expression in wild-type seedlings and reduced RNA editing of matK-706, accD-794, ndhD-383 and ndhF-290 in the morf9 mutant. RNA editing efficiency of ndhD-383 and ndhF-290 sites was diminished in the gin2/morf9 double mutants, and that of matK-706, accD-794, ndhD-383 and ndhF-290 sites were significantly diminished in the snrk1/morf9 double mutants. In contrast, overexpressing HXK1 or SnRK1 promoted RNA editing rate of matK-706, accD-794, ndhD-383and ndhF-290 in leaves of morf9 mutants, suggesting that HXK1 partially impacts MORF9 mediated ndhD-383 and ndhF-290 editing, while SnRK1 may only affect MORF9-mediated ndhF-290 site editing. Collectively, these findings suggest that sugar and/or its intermediary metabolites impair MORF9-dependent plastid RNA editing resulting in derangements of plant root development.

Natural Albino Mutant of Daylily (Hemerocallis spp.) Reveals a Link between Drought Sensitivity and Photosynthetic Pigments Metabolism.

Mutant analysis remains one of the main genetic tools for characterising unclarified gene functions in plants, especially in non-model plants. Daylily (Hemerocallis spp.) is a popular perennial ornamental plant grown worldwide. Analysis of daylily mutants can enhance understanding of genes regulating the albino phenotype and improve the cultivar quality of daylily. The natural albino mutant (Alb-⁣/-) was isolated by screening a self-pollinated progeny of daylily cultivar 'black-eyed stella'. Transmission electron microscopy was used in analysing the structure of plastids between mutant and wild-type seedlings. The content of chlorophyll, carotenoids and chlorophyll precursors in plants was measured by ultraviolet spectrophotometry. RNA sequencing and physiological measurements were performed to explore the association between drought tolerance and mutation. All the seedlings of the daylily albino mutants died spontaneously within fifteen days after germination when grown in soil. The carotenoid and chlorophyll content in the leaves of the mutant plants significantly decreased compared with those of the wild-type control. The mutant plants displayed stunted growth, and their leaves were white or light yellow in color. Abnormal plastids such as those showing endomembrane vesiculation and lacking stacking were discovered in the leaves of mutant plants. Furthermore, genetic analysis revealed that a single recessive nuclear gene mutation led to the albino trait, RNA sequencing and real-time quantitative PCR validation showed extensive differences in gene expression between the mutant plants and the wild-type control, and most of the genes related to chlorophyll metabolism were down-regulated, with foldchange ranging from 0.20-0.49. Additionally, the surviving homozygous plants (Alb+⁣/+), which do not contain this mutation, were also isolated by analysing the phenotype of their self-pollinated progeny. The net photosynthesis rate and light saturation point of Alb+⁣/+ were higher than those of heterozygous (Alb+⁣/-) plants. Additionally, the Alb+⁣/+ plants were more tolerant to drought conditions than the Alb+⁣/- plants, suggesting that a heterozygous Alb- mutation is sufficient to negatively affect photosynthetic efficiency and drought tolerance. The albino mutation negatively affects photosynthetic efficiency and drought tolerance, and homozygous mutation is required for the characteristic albino phenotype. This work highlights the link between albino mutation, photosynthetic pigment metabolism and drought sensitivity in daylily.

Fusarium Induced Anatomical and Biochemical Alterations in Wild Type and DPA-treated Wheat Seedlings

Wheat (Triticum aestivum) employs various strategies to defend against Fusarium oxysporum, a soil-borne vascular fungal pathogen that disrupts structural integrity and metabolism. The purpose of this research was to ascertain the alterations of anatomical and biochemical responses in wild-type (WT) and DPA-treated wheat (T. aestivum) seedlings exposed to F. oxysporum. The WT and DPA-treated seedlings showed disorganization of parenchyma cells, sclerenchyma cells, vascular bundles (VBs), and lower numbers of xylem (Xy) and phloem (Ph) cells, and reduced thickness of the cuticle layer (C) at the epidermal layer of shoots. The content of chlorophyll (Chl), carbohydrate, and nucleic acid was reduced in WT and DPA-treated seedlings during infection. Enhanced defense responses through peroxidase (POD), and polyphenol oxidase (PPO) was observed to be high in WT as compared to DPA-treated seedlings under stress condition. In addition, the content of salicylic acid (SA) and phenolics was increased in WT than DPA under stress condition. However, the DPA-treated seedlings showed enhanced growth of fungal mycelia compared to WT during stress condition. Hence, the anatomical and biochemical aspects of DPA-treated seedlings decreased as compared to WT when exposed to F. oxysporum.