Crucial role of SWL1 in chloroplast biogenesis and development in Arabidopsis thaliana.

KEY MESSAGE: The disruption of the SWL1 gene leads to a significant down regulation of chloroplast and secondary metabolites gene expression in Arabidopsis thaliana. And finally results in a dysfunction of chloroplast and plant growth. Although the development of the chloroplast has been a consistent focus of research, the corresponding regulatory mechanisms remain unidentified. In this study, the CRISPR/Cas9 system was used to mutate the SWL1 gene, resulting in albino cotyledons and variegated true leaf phenotype. Confocal microscopy and western blot of chloroplast protein fractions revealed that SWL1 localized in the chloroplast stroma. Electron microscopy indicated chloroplasts in the cotyledons of swl1 lack well-defined grana and internal membrane structures, and similar structures have been detected in the albino region of variegated true leaves. Transcriptome analysis revealed that down regulation of chloroplast and nuclear gene expression related to chloroplast, including light harvesting complexes, porphyrin, chlorophyll metabolism and carbon metabolism in the swl1 compared to wild-type plant. In addition, proteomic analysis combined with western blot analysis, showed that a significant decrease in chloroplast proteins of swl1. Furthermore, the expression of genes associated with secondary metabolites and growth hormones was also reduced, which may be attributed to SWL1 associated with absorption and fixation of inorganic carbon during chloroplast development. Together, the above findings provide valuable information to elucidate the exact function of SWL1 in chloroplast biogenesis and development.

Genome-Wide Association Study Uncovers Genomic Regions Associated with Coleoptile Length in a Worldwide Collection of Oat.

The length of coleoptile is crucial for determining the sowing depth of oats in low-precipitation regions, which is significant for oat breeding programs. In this study, a diverse panel of 243 oat accessions was used to explore coleoptile length in two independent experiments. The panel exhibited significant variation in coleoptile length, ranging from 4.66 to 8.76 cm. Accessions from Africa, America, and the Mediterranean region displayed longer coleoptile lengths than those from Asia and Europe. Genome-wide association studies (GWASs) using 26,196 SNPs identified 34 SNPs, representing 32 quantitative trait loci (QTLs) significantly associated with coleoptile length. Among these QTLs, six were consistently detected in both experiments, explaining 6.43% to 10.07% of the phenotypic variation. The favorable alleles at these stable loci additively increased coleoptile length, offering insights for pyramid breeding. Gene Ontology (GO) analysis of the 350 candidate genes underlying the six stable QTLs revealed significant enrichment in cell development-related processes. Several phytochrome-related genes, including auxin transporter-like protein 1 and cytochrome P450 proteins, were found within these QTLs. Further validation of these loci will enhance our understanding of coleoptile length regulation. This study provides new insights into the genetic architecture of coleoptile length in oats.

Role of sugars in the apical hook development of Arabidopsis etiolated seedlings.

KEY MESSAGE: The sugar supply in the medium affects the apical hook development of Arabidopsis etiolated seedlings. In addition, we provided the mechanism insights of this process. Dicotyledonous plants form an apical hook structure to shield their young cotyledons from mechanical damage as they emerge from the rough soil. Our findings indicate that sugar molecules, such as sucrose and glucose, are crucial for apical hook development. The presence of sucrose and glucose allows the apical hooks to be maintained for a longer period compared to those grown in sugar-free conditions, and this effect is dose-dependent. Key roles in apical hook development are played by several sugar metabolism pathways, including oxidative phosphorylation and glycolysis. RNA-seq data revealed an up-regulation of genes involved in starch and sucrose metabolism in plants grown in sugar-free conditions, while genes associated with phenylpropanoid metabolism were down-regulated. This study underscores the significant role of sugar metabolism in the apical hook development of etiolated Arabidopsis seedlings.

DEFECTIVELY ORGANIZED TRIBUTARIES 5 is not required for leaf venation patterning in Arabidopsis thaliana.

The search for genetic regulators of leaf venation patterning started over 30 years ago, primarily focused on mutant screens in the eudicotyledon Arabidopsis thaliana. Developmental perturbations in either cotyledons or true leaves led to the identification of transcription factors required to elaborate the characteristic reticulated vein network. An ortholog of one of these, the C2H2 zinc finger protein DEFECTIVELY ORGANIZED TRIBUTARIES 5 (AtDOT5), was recently identified through transcriptomics as a candidate regulator of parallel venation in maize (Zea mays) leaves. To elucidate how AtDOT5 regulates vein patterning, we generated three independent loss-of-function mutations by gene editing in Arabidopsis. Surprisingly, none of them exhibited any obvious phenotypic perturbations. To reconcile our findings with earlier reports, we re-evaluated the original Atdot5-1 and Atdot5-2 alleles. By genome sequencing, we show that reported mutations at the Atdot5-1 locus are actually polymorphisms between Landsberg erecta and Columbia ecotypes, and that other mutations present in the background most likely cause the pleiotropic mutant phenotype observed. We further show that a T-DNA insertion in the Atdot5-2 locus has no impact on leaf venation patterns when segregated from other T-DNA insertions present in the original line. We thus conclude that AtDOT5 plays no role in leaf venation patterning in Arabidopsis.

LEAFY COTYLEDON 2: A Regulatory Factor of Plant Growth and Seed Development.

Transcription factors are key molecules in the regulation of gene expression in all organisms. The transcription factor LEAFY COTYLEDON 2 (LEC2), which belongs to the DNA-binding protein family, contains a B3 domain. The transcription factor is involved in the regulation of important plant biological processes such as embryogenesis, somatic embryo formation, seed storage protein synthesis, fatty acid metabolism, and other important biological processes. Recent studies have shown that LEC2 regulates the formation of lateral roots and influences the embryonic resetting of the parental vernalization state. The orthologs of LEC2 and their regulatory effects have also been identified in some crops; however, their regulatory mechanism requires further investigation. Here, we summarize the most recent findings concerning the effects of LEC2 on plant growth and seed development. In addition, we discuss the potential molecular mechanisms of the action of the LEC2 gene during plant development.

B-box protein BBX32 integrates light and brassinosteroid signals to inhibit cotyledon opening.

Cotyledon opening is a key morphological change that occurs in seedlings during de-etiolation. Brassinosteroids (BRs) inhibit the opening of cotyledons in darkness while light promotes cotyledon opening. The molecular regulation of the interplay between light and BR to regulate cotyledon opening is not well understood. Here, we show the B-box protein BBX32 negatively regulates light signaling and promotes BR signaling to inhibit cotyledon opening in Arabidopsis (Arabidopsis thaliana). BBX32 is highly expressed in the cotyledons of seedlings during de-etiolation. bbx32 and 35S:BBX32 seedlings exhibit enhanced and reduced cotyledon opening, respectively, in response to both light and brassinazole treatment in dark, suggesting that BBX32 mediates cotyledon opening through both light and BR signaling pathways. BBX32 expression is induced by exogenous BR and is upregulated in bzr1-1D (BRASSINAZOLE RESISTANT1-1D). Our in vitro and in vivo interaction studies suggest that BBX32 physically interacts with BZR1. Further, we found that PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) interacts with BBX32 and promotes BR-mediated cotyledon closure. BBX32, BZR1, and PIF3 regulate the expression of common target genes that modulate the opening and closing of cotyledons. Our work suggests BBX32 integrates light and BR signals to regulate cotyledon opening during de-etiolation.

Over-Expression of the Cell-Cycle Gene LaCDKB1;2 Promotes Cell Proliferation and the Formation of Normal Cotyledonary Embryos during Larix kaempferi Somatic Embryogenesis.

Somatic embryogenesis is an effective tool for the production of forest tree seedlings with desirable characteristics; however, the low initiation frequency and productivity of high-quality mature somatic embryos are still limiting factors for Larix kaempferi (Japanese larch). Here, we analyzed the expression pattern of L. kaempferi cyclin-dependent kinase B 1;2 (LaCDKB1;2) during somatic embryogenesis in L. kaempferi and its relationship with the cell proliferation rate. We also analyzed the effect of LaCDKB1;2 over-expression on somatic embryo quality. The results revealed a positive correlation between LaCDKB1;2 expression and the cell proliferation rate during the proliferation stage. After LaCDKB1;2 over-expression, the proliferation rate of cultures increased, and the number of somatic embryos in transgenic cultures was 2.69 times that in non-transformed cultures. Notably, the number of normal cotyledonary embryos in transgenic cultures was 3 times that in non-transformed cultures, indicating that LaCDKB1;2 not only increases the proliferation of cultures and the number of somatic embryos but also improves the quality of somatic embryos. These results provide insight into the regulatory mechanisms of somatic embryogenesis as well as new Larix breeding material.

Exogenous carbon source supplementation counteracts root and hypocotyl growth limitations under increased cotyledon shading, with glucose and sucrose differentially modulating growth curves.

Plant growth is continuously modulated by endogenous and exogenous stimuli. By no means the only, but well described, signaling molecules produced in plants and distributed through the plant body to orchestrate efficient growth are photosynthates. Light is a potent exogenous stimulus that determines, first, the rate of photosynthesis, but also the rate of plant growth. Root meristem activity is reduced with direct illumination but enhanced with increased sugar levels. With reduced cotyledon illumination, the seedling increases hypocotyl elongation until adequate light exposure is again provided. If endogenous carbon sources are limited, this leads to a temporary inhibition of root growth. Experimental growth conditions include exogenous supplementation of sucrose or glucose in addition to culturing seedlings under light exposure in Petri dishes. We compared total root length and hypocotyl elongation of Arabidopsis thaliana wild type Col-0 in response to illumination status and carbon source in the growth medium. Overall, sucrose supplementation promoted hypocotyl and root length to a greater extent than glucose supplementation. Glucose promoted root length compared to non-supplemented seedlings especially when cotyledon illumination was greatly reduced.

24-epibrassinolide confers tolerance against deep-seeding stress in Zea mays L. coleoptile development by phytohormones signaling transduction and their interaction network.

Coleoptile/mesocotyl elongation influence seedling emergence and establishment, is major causes of maize deep-seeding tolerance (DST). Detailed analyses on molecular basis underlying their elongation mediated by brassinosteroid under deep-seeding stress (DSS) could provide meaningful information for key factors controlling their elongation. Here we monitored transcriptome and phytohormones changes specifically in elongating coleoptile/mesocotyl in response to DSS and 24-epibrassinolide (EBR)-signaling. Phenotypically, contrasting maize evolved variant organs to positively respond to DST, longer coleoptile/mesocoty of K12/W64A was a desirable organ for seedling under DSS. Applied-EBR improved maize DST, and their coleoptiles/mesocotyls were further elongated. 15,607/20,491 differentially expressed genes (DEGs) were identified in W64A/K12 coleoptile, KEGG analysis showed plant hormone signal transduction, starch and sucrose metabolism, valine, leucine, and isoleucine degradation were critical processes of coleoptile elongation under DSS and EBR signaling, further highly interconnected network maps including 79/142 DEGs for phytohormones were generated. Consistent with these DEGs expression, interactions, and transport, IAA, GA3, ABA, and Cis-ZT were significantly reduced while EBR, Trans-ZT, JA, and SA were clearly increased in coleoptile under DSS and EBR-signaling. These results enrich our knowledge about the genes and phytohormones regulating coleoptile elongation in maize, and help improve future studies on corresponding genes and develop varieties with DST.

PPR protein Early Chloroplast Development 2 is essential for chloroplast development at the early stage of Arabidopsis development.

Chloroplast biogenesis and development regulation have long been a focus of research; however, the underlying mechanisms of these processes have not yet been fully elucidated. Pentatricopeptide repeat (PPR) proteins have been shown to play key roles in chloroplast development. Here, we identified a novel P-type PPR protein, Early Chloroplast Development 2 (ECD2), and the ecd2 mutant resulted in embryo lethality. The RNAi lines of ECD2 showed varying degrees of albino cotyledons and abnormal chloroplast development, but true leaves were similar to the wild-type. Further analysis revealed that ECD2 was responsible for chloroplast gene expression and group II intron splicing of several genes. Transcriptome analysis combined with quantitative real-time PCR showed that ECD2 was associated with the expression of ribosomal genes and accumulation of chloroplast ribosomes. Overall, our results indicate that ECD2 is critically important for early chloroplast development in cotyledon.

Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives

BACKGROUND: In order to produce an effective callus in Echinacea purpurea L.; determination of the explant type and growth regulators that best respond to callus induction and the optimization of the culture conditions to increase the amount of caffeic acid derivatives (CADs) in the obtained callus. CADs contents of callus cultures of E. purpurea were evaluated by establishing an effective callus induction system in vitro. RESULTS: Various medium containing different growth regulators were tested using leaf, petiole, cotyledon and root as the explants. The best callus development was achieved in MS medium with 1.0 mg l 1 2,4- D + 2.0 mg l 1 BAP in leaf, 1.0 mg l 1 NAA + 0.5 mg l 1 TDZ in petiole, 2.0 mg l 1 NAA + 1.0 mg l 1 TDZ in cotyledon and 0.5 mg l 1 NAA + 0.5 mg l 1 BAP in roots. Upon optimisation of callus growth, each type of explant was cultured for 4, 6, 8 and 10 weeks in medium for the analyses of caftaric acid, chlorogenic acid, caffeic acid and chicoric acid contents. The highest amounts of caftaric acid (4.11 mg/g) and chicoric acid (57.89 mg/g) were found from petiole explants and chlorogenic acid (8.83 mg/g) from root explants at the end of the 10-week culture time. CONCLUSIONS: As a result of the present study, the production of caffeic acid derivatives was performed by providing the optimization of E. purpurea L. callus cultures. Effective and repeatable protocols established in this study may offer help for further studies investigating the production of caffeic acid derivatives in vitro.

Temporal changes in metabolism late in seed development affect biomass composition.

The negative association between protein and oil production in soybean (Glycine max) seed is well-documented. However, this inverse relationship is based primarily on the composition of mature seed, which reflects the cumulative result of events over the course of soybean seed development and therefore does not convey information specific to metabolic fluctuations during developmental growth regimes. In this study, we assessed maternal nutrient supply via measurement of seed coat exudates and metabolite levels within the cotyledon throughout development to identify trends in the accumulation of central carbon and nitrogen metabolic intermediates. Active metabolic activity during late seed development was probed through transient labeling with 13C substrates. The results indicated: (1) a drop in lipid contents during seed maturation with a concomitant increase in carbohydrates, (2) a transition from seed filling to maturation phases characterized by quantitatively balanced changes in carbon use and CO2 release, (3) changes in measured carbon and nitrogen resources supplied maternally throughout development, (4) 13C metabolite production through gluconeogenic steps for sustained carbohydrate accumulation as the maternal nutrient supply diminishes, and (5) oligosaccharide biosynthesis within the seed coat during the maturation phase. These results highlight temporal engineering targets for altering final biomass composition to increase the value of soybeans and a path to breaking the inverse correlation between seed protein and oil content.

Phenotypic Study of Photomorphogenesis in Arabidopsis Seedlings.

Light is one of the most important environmental factors, serving as the energy source of photosynthesis and a cue for plant developmental programs, called photomorphogenesis. Here, we provide a standardized operation to measure physiological parameters of photomorphogenesis, including in hypocotyl length, cotyledon size, and anthocyanin content.

Experimental Procedures for Studying Skotomorphogenesis in Arabidopsis thaliana.

Seedlings grown in darkness exhibit distinct morphologies comparing with light-grown seedlings. Elongated hypocotyls, closed yellow cotyledons, and the formation of apical hooks are typical characteristics for etiolated seedlings, which are collectively named skotomorphogenesis. Various plant hormones and environmental factors are essential for maintaining skotomorphogenesis. Due to the diverse morphological outcomes in etiolated seedlings grown under different treatments, studies on skotomorphogenesis are of particular importance to reveal the molecular mechanisms underlying plant response to environmental cues. Here, we detailed experimental procedures to facilitate researchers who are investigating etiolation growth-related studies.

The Arabidopsis STE20/Hippo kinase SIK1 regulates polarity independently of PIN proteins.

Polarity is a feature of life. In higher plants, non-autonomous polarity is largely directed by auxin, the morphogen that drives its own polarized flow, Polar Auxin Transport (PAT), to guide patterning events such as phyllotaxis and tropism. The plasma membrane-localized PIN-FORMED (PIN) auxin efflux carriers are rate-limiting factors in PAT. In yeasts and metazoans, the STE20 kinases are key players in cell polarity. We had previously characterized SIK1 as a STE20/Hippo orthologue in Arabidopsis and confirmed its function in mitotic exit and organ growth. Here we explore the possible link between SIK1, auxin, PIN, and polarity. Abnormal phyllotaxis and gravitropism were observed in sik1. sik1 was more sensitive to exogenous auxin in primary root elongation and lateral root emergence. RNA-Seq revealed reduced expression in auxin biosynthesis genes and induced expression of auxin flux carriers in sik1. However, normal tissue- and sub-cellular localization patterns of PIN1 and PIN2 were observed in sik1. The dark-induced vacuolar degradation of PIN2 also appeared normal in sik1. An additive phenotype was observed in the sik1 pin1 double mutant, indicating that SIK1 does not directly regulate PIN1. The polarity defects of sik1 are hence unlikely mediated by PINs and await future exploration.

Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants.

Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.

Metabolomics Analyses of Cotyledon and Plumule Showing the Potential Domestic Selection in Lotus Breeding.

Lotus (Nelumbo nucifera) seeds are widely consumed as functional food or herbal medicine, of which cotyledon (CL) is the main edible part, and lotus plumule (LP) is commonly utilized in traditional Chinese medicine. However, few studies have been conducted to investigate the chemical components of CL and LP in dry lotus seeds, not to mention the comparison between wild and domesticated varieties. In this study, a widely targeted metabolomics approach based on Ultra Performance Liquid Chromatography-electrospray ionization-Tandem mass spectrometry (UPLC-ESI-MS/MS) was utilized to analyze the metabolites in CL and LP of China Antique ("CA", a wild variety) and Jianxuan-17 ("JX", a popular cultivar). A total of 402 metabolites were identified, which included flavonoids (23.08% to 27.84%), amino acids and derivatives (14.18-16.57%), phenolic acids (11.49-12.63%), and lipids (9.14-10.95%). These metabolites were classified into ten clusters based on their organ or cultivar-specific characters. Most of these metabolites were more abundant in LP than in CL for both varieties, except for metabolites belonging to organic acids and lipids. The analysis of differentially accumulated metabolites (DAMs) demonstrated that more than 25% of metabolites detected in our study were DAMs in CL and LP comparing "JX" with "CA", most of which were less abundant in "JX", including 35 flavonoids in LP, 23 amino acids and derivatives in CL, 7 alkaloids in CL, and 10 nucleotides and derivatives in LP, whereas all of 11 differentially accumulated lipids in LP were more abundant in "JX". Together with the fact that the seed yield of "JX" is much higher than that of "CA", these results indicated that abundant metabolites, especially the functional secondary metabolites (mainly flavonoids and alkaloids), were lost during the process of breeding selection.

TCP transcription factors suppress cotyledon trichomes by impeding a cell differentiation-regulating complex.

Trichomes are specialized epidermal cells that act as barriers against biotic and abiotic stresses. Although the formation of trichomes on hairy organs is well studied, the molecular mechanisms of trichome inhibition on smooth organs are still largely unknown. Here, we demonstrate that the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors inhibit the formation of trichomes on cotyledons in Arabidopsis (Arabidopsis thaliana). The tcp2/3/4/5/10/13/17 septuple mutant produces cotyledons with ectopic trichomes on the adaxial sides. The expression patterns of TCP genes are developmentally regulated during cotyledon development. TCP proteins directly interact with GLABRA3 (GL3), a key component of the MYB transcription factor/basic helix-loop-helix domain protein/WD40-repeat proteins (MYB-bHLH-WD40, MBW) complex essential for trichome formation, to interfere with the transactivation activity of the MBW complex in cotyledons. TCPs also disrupt the MBW complex-R3 MYB negative feedback loop by directly promoting the expression of R3 MYB genes, which enhance the repression of the MBW complex. Our findings reveal a molecular framework in which TCPs suppress trichome formation on adaxial sides of cotyledons by repressing the activity of the MBW complex at the protein level and the transcripts of R3 MYB genes at the transcriptional level.

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production.

Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T1 and T2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.