Arabidopsis Seedling Development Research Articles

Knockout of endoplasmic reticulum-localized molecular chaperone HSP90.7 impairs seedling development and cellular auxin homeostasis in Arabidopsis.

The Arabidopsis endoplasmic reticulum-localized heat shock protein HSP90.7 modulates tissue differentiation and stress responses; however, complete knockout lines have not been previously reported. In this study, we identified and analyzed a mutant allele, hsp90.7-1, which was unable to accumulate the HSP90.7 full-length protein and showed seedling lethality. Microscopic analyses revealed its essential role in male and female fertility, trichomes and root hair development, proper chloroplast function, and apical meristem maintenance and differentiation. Comparative transcriptome and proteome analyses also revealed the role of the protein in a multitude of cellular processes. Particularly, the auxin-responsive pathway was specifically downregulated in the hsp90.7-1 mutant seedlings. We measured a much-reduced auxin content in both root and shoot tissues. Through comprehensive histological and molecular analyses, we confirmed PIN1 and PIN5 accumulations were dependent on the HSP90 function, and the TAA-YUCCA primary auxin biosynthesis pathway was also downregulated in the mutant seedlings. This study therefore not only fulfilled a gap in understanding the essential role of HSP90 paralogs in eukaryotes but also provided a mechanistic insight on the ER-localized chaperone in regulating plant growth and development via modulating cellular auxin homeostasis.

GLK transcription factors accompany ELONGATED HYPOCOTYL5 to orchestrate light-induced seedling development in Arabidopsis.

Light-induced de-etiolation is an important aspect of seedling photomorphogenesis. GOLDEN2 LIKE (GLK) transcriptional regulators are involved in chloroplast development, but to what extent they participate in photomorphogenesis is not clear. Here, we show that ELONGATED HYPOCOTYL5 (HY5) binds to GLK promoters to activate their expression, and also interacts with GLK proteins in Arabidopsis (Arabidopsis thaliana). The chlorophyll content in the de-etiolating Arabidopsis seedlings of the hy5 glk2 double mutants was lower than that in the hy5 single mutant. GLKs inhibited hypocotyl elongation, and the phenotype could superimpose on the hy5 phenotype. Correspondingly, GLK2 regulated the expression of photosynthesis and cell elongation genes partially independent of HY5. Before exposure to light, DE-ETIOLATED 1 (DET1) affected accumulation of GLK proteins. The enhanced etioplast development and photosystem gene expression observed in the det1 mutant were attenuated in the det1 glk2 double mutant. Our study reveals that GLKs act downstream of HY5, or additive to HY5, and are likely quantitatively adjusted by DET1, to orchestrate multiple developmental traits during the light-induced skotomorphogenesis-to-photomorphogenesis transition in Arabidopsis.

Vacuolar Sugar Transporter TMT2 Plays Crucial Roles in Germination and Seedling Development in Arabidopsis

Vacuolar sugar transporters transport sugar across the tonoplast, are major players in maintaining sugar homeostasis, and therefore play vital roles in plant growth, development, and biomass yield. In this study, we analyzed the physiological roles of the tonoplast monosaccharide transporter 2 (TMT2) in Arabidopsis. In contrast to the wild type (WT) that produced uniform seedlings, the tmt2 mutant produced three types of offspring: un-germinated seeds (UnG), seedlings that cannot form true leaves (tmt2-S), and seedlings that develop normally (tmt2-L). Sucrose, glucose, and fructose can substantially, but not completely, rescue the abnormal phenotypes of the tmt2 mutant. Abnormal cotyledon development, arrested true leaf development, and abnormal development of shoot apical meristem (SAM) were observed in tmt2-S seedlings. Cotyledons from the WT and tmt2-L seedlings restored the growth of tmt2-S seedlings through micrografting. Moreover, exogenous sugar sustained normal growth of tmt2-S seedlings with cotyledon removed. Finally, we found that the TMT2 deficiency resulted in growth defects, most likely via changing auxin signaling, target of rapamycin (TOR) pathways, and cellular nutrients. This study unveiled the essential functions of TMT2 for seed germination and initial seedling development, ensuring cotyledon function and mobilizing sugars from cotyledons to seedlings. It also expanded the current knowledge on sugar metabolism and signaling. These findings have fundamental implications for enhancing plant biomass production or seed yield in future agriculture.

MKKK20 works as an upstream triple-kinase of MKK3-MPK6-MYC2 module in Arabidopsis seedling development

The mitogen-activated protein kinase (MAPK) cascade is involved in several signal transduction processes in eukaryotes. Here, we report a mechanistic function of MAP kinase kinase kinase 20 (MKKK20) in light signal transduction pathways. We show that MKKK20 acts as a negative regulator of photomorphogenic growth at various wavelengths of light. MKKK20 not only regulates the expression of light signaling pathway regulatory genes but also gets regulated by the same pathway genes. The atmyc2 mkkk20 double mutant analysis shows that MYC2 works downstream to MKKK20 in the regulation of photomorphogenic growth. MYC2 directly binds to the promoter of MKKK20 to modulate its expression. The protein-protein interaction study indicates that MKKK20 physically interacts with MYC2, and this interaction likely suppresses the MYC2-mediated promotion of MKKK20 expression. Further, the protein phosphorylation studies demonstrate that MKKK20 works as the upstream kinase of MKK3-MPK6-MYC2 module in photomorphogenesis.

ABA signaling prevents phosphodegradation of the SR45 splicing factor to alleviate inhibition of early seedling development in Arabidopsis

Serine/arginine-rich (SR) proteins are conserved splicing regulators that play important roles in plant stress responses, namely those mediated by the abscisic acid (ABA) hormone. The Arabidopsis thaliana SR-like protein SR45 is a described negative regulator of the ABA pathway during early seedling development. How the inhibition of growth by ABA signaling is counteracted to maintain plant development under stress conditions remains largely unknown. Here, we show that SR45 overexpression reduces Arabidopsis sensitivity to ABA during early seedling development. Biochemical and confocal microscopy analyses of transgenic plants expressing fluorescently tagged SR45 revealed that exposure to ABA dephosphorylates the protein at multiple amino acid residues and leads to its accumulation, due to SR45 stabilization via reduced ubiquitination and proteasomal degradation. Using phosphomutant and phosphomimetic transgenic Arabidopsis lines, we demonstrate the functional relevance of ABA-mediated dephosphorylation of a single SR45 residue, T264, in antagonizing SR45 ubiquitination and degradation to promote its function as a repressor of seedling ABA sensitivity. Our results reveal a mechanism that negatively autoregulates ABA signaling and allows early plant growth under stress via posttranslational control of the SR45 splicing factor.

The conjugation of SUMO to the transcription factor MYC2 functions in blue light-mediated seedling development in Arabidopsis.

A key function of photoreceptor signaling is the coordinated regulation of a large number of genes to optimize plant growth and development. The basic helix loop helix (bHLH) transcription factor MYC2 is crucial for regulating gene expression in Arabidopsis thaliana during development in blue light. Here we demonstrate that blue light induces the SUMOylation of MYC2. Non-SUMOylatable MYC2 is less effective in suppressing blue light-mediated photomorphogenesis than wild-type (WT) MYC2. MYC2 interacts physically with the SUMO proteases SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2. Blue light exposure promotes the degradation of SPF1 and SPF2 and enhances the SUMOylation of MYC2. Phenotypic analysis revealed that SPF1/SPF2 function redundantly as positive regulators of blue light-mediated photomorphogenesis. Our data demonstrate that SUMO conjugation does not affect the dimerization of MYC transcription factors but modulates the interaction of MYC2 with its cognate DNA cis-element and with the ubiquitin ligase Plant U-box 10 (PUB10). Finally, we show that non-SUMOylatable MYC2 is less stable and interacts more strongly with PUB10 than the WT. Taken together, we conclude that SUMO functions as a counterpoint to the ubiquitin-mediated degradation of MYC2, thereby enhancing its function in blue light signaling.

The photomorphogenic repressors BBX28 and BBX29 integrate light and brassinosteroid signaling to inhibit seedling development in Arabidopsis.

B-box containing proteins (BBXs) integrate light and various hormonal signals to regulate plant growth and development. Here, we demonstrate that the photomorphogenic repressors BBX28 and BBX29 positively regulate brassinosteroid (BR) signaling in Arabidopsis thaliana seedlings. Treatment with the BR brassinolide stabilized BBX28 and BBX29, which partially depended on BR INSENSITIVE1 (BRI1) and BIN2. bbx28 bbx29 seedlings exhibited larger cotyledon aperture than the wild-type when treated with brassinazole in the dark, which partially suppressed the closed cotyledons of brassinazole resistant 1-1D (bzr1-1D). Consistently, overexpressing BBX28 and BBX29 partially rescued the short hypocotyls of bri1-5 and bin2-1 in both the dark and light, while the loss-of-function of BBX28 and BBX29 partially suppressed the long hypocotyls of bzr1-1D in the light. BBX28 and BBX29 physically interacted with BR-ENHANCED EXPRESSION1 (BEE1), BEE2, and BEE3 and enhanced their binding to and activation of their target genes. Moreover, BBX28 and BBX29 as well as BEE1, BEE2, and BEE3 increased BZR1 accumulation to promote the BR signaling pathway. Therefore, both BBX28 and BBX29 interact with BEE1, BEE2, and BEE3 to orchestrate light and BR signaling by facilitating the transcriptional activity of BEE target genes. Our study provides insights into the pivotal roles of BBX28 and BBX29 as signal integrators in ensuring normal seedling development.

Non-TZF Transcriptional Activator AtC3H12 Negatively Affects Seed Germination and Seedling Development in Arabidopsis.

CCCH zinc finger proteins are a large protein family and are classified as either tandem CCCH zinc finger (TZF) or non-TZF proteins. The roles of TZF genes in several plants have been well determined, whereas the functions of many non-TZF genes in plants remain uncharacterized. Herein, we describe biological and molecular functions of AtC3H12, an Arabidopsis non-TZF protein containing three CCCH zinc finger motifs. AtC3H12 has orthologs in several plant species but has no paralog in Arabidopsis. AtC3H12-overexpressing transgenic plants (OXs) germinated slower than wild-type (WT) plants, whereas atc3h12 mutants germinated faster than WT plants. The fresh weight (FW) and primary root lengths of AtC3H12 OX seedlings were lighter and shorter than those of WT seedlings, respectively. In contrast, FW and primary root lengths of atc3h12 seedlings were heavier and longer than those of WT seedlings, respectively. AtC3H12 was localized in the nucleus and displayed transactivation activity in both yeast and Arabidopsis. We found that the 97–197 aa region of AtC3H12 is an important part for its transactivation activity. Detection of expression levels and analysis of Arabidopsis transgenic plants harboring a PAtC3H12::GUS construct showed that AtC3H12 expression increases as the Arabidopsis seedlings develop. Taken together, our results demonstrate that AtC3H12 negatively affects seed germination and seedling development as a nuclear transcriptional activator in Arabidopsis. To our knowledge, this is the first report to show that non-TZF proteins negatively affect plant development as nuclear transcriptional activators.

A dual-specificity phosphatase, MAP kinase phosphatase 1, positively regulates blue light-mediated seedling development in Arabidopsis.

A dual-specificity phosphatase MKP1 negatively regulates the activity of MPK6 by dephosphorylating it and acts as a positive regulator of blue light (BL)-mediated photomorphogenic development in Arabidopsis. Reversible phosphorylation of proteins is one of the major post-translational modifications in nearly all signaling pathways in plants. MAP kinase phosphatases are very crucial in the regulation of MAPKs as they dephosphorylate both threonine (Thr) and tyrosine (Tyr) residues within the T-X-Y motif of active MAPKs. Therefore, to gain insight of involvement of MAP kinase phosphatases in the regulation of light signaling, we searched for the potential phosphatase which may regulate the function of MPK6, a negative regulator of blue light (BL)-mediated photomorphogenic development. We report here the identification of a dual-specificity phosphatase, MAP kinase phosphatase 1 (MKP1) as a positive regulator of BL-mediated seedling development. Overexpression of MKP1 enhances the BL-induced inhibition of hypocotyl elongation and displays more opened cotyledons. We also show that MKP1OE accumulates more pigments and positively affects the expression of downstream light-related genes in response to BL. In vitro and in vivo evidences also demonstrate that MKP1 not only interacts with but also dephosphorylates MPK6 in BL. In addition, MKP1 regulates stability as well as activity of MPK6 upon BL. Taken together our study highlights the important role of phosphatases in the regulation of a signaling pathway and identifies the role of MKP1 in the negative regulation of MPK6 activity leading to a change in BL-induced photomorphogenic responses.

JMJ17-WRKY40 and HY5-ABI5 modules regulate the expression of ABA-responsive genes in Arabidopsis.

Abscisic acid (ABA) plays a crucial role in the adaptation of young seedlings to environmental stresses. However, the role of epigenetic components and core transcriptional machineries in the effect of ABA on seed germination and seedling growth remain unclear. Here, we show that a histone 3 lysine 4 (H3K4) demethylase, JMJ17, regulates the expression of ABA-responsive genes during seed germination and seedling growth. Using comparative interactomics, WRKY40, a central transcriptional repressor in ABA signaling, was shown to interact with JMJ17. WRKY40 facilitates the recruitment of JMJ17 to the ABI5 chromatin, which removes gene activation marks (H3K4me3) from the ABI5 chromatin, thereby repressing its expression. Additionally, WRKY40 represses the transcriptional activation activity of HY5, which can activate ABI5 expression by directly binding to its promoter. An increase in ABA concentrations decreases the affinity of WRKY40 for the ABI5 promoter. Thus, WRKY40 and JMJ17 are released from the ABI5 chromatin, activating HY5. The accumulated ABI5 protein further shows heteromeric interaction with HY5, and thus synergistically activates its own expression. Our findings reveal a novel transcriptional switch, composed of JMJ17-WRKY40 and HY5-ABI5 modules, which regulates the ABA response during seed germination and seedling development in Arabidopsis.

HY5 and phytochrome activity modulate shoot-to-root coordination during thermomorphogenesis in Arabidopsis

Temperature is one of the most impactful environmental factors to which plants adjust their growth and development. Although the regulation of temperature signaling has been extensively investigated for the aerial part of plants, much less is known and understood about how roots sense and modulate their growth in response to fluctuating temperatures. Here, we found that shoot and root growth responses to high ambient temperature are coordinated during early seedling development in Arabidopsis A shoot signaling module that includes HY5, the phytochromes and the PIFs exerts a central function in coupling these growth responses and maintaining auxin levels in the root. In addition to the HY5/PIF-dependent shoot module, a regulatory axis composed of auxin biosynthesis and auxin perception factors controls root responses to high ambient temperature. Taken together, our findings show that shoot and root developmental responses to temperature are tightly coupled during thermomorphogenesis and suggest that roots integrate energy signals with local hormonal inputs.

The Z-box binding factors (ZBFs): emerging new facets in Arabidopsis seedling development

The transcriptional regulatory networks play important roles in Arabidopsis seedling development. It is well established that large-scale transcriptional reprogramming occurs during transition between skotomorphogenesis and photomorphogenesis. ZBFs (Z-box Binding Factors), represented by a set of genes, ZBF1 (encoding ZBF1/MYC2 bHLH protein), ZBF2 (encoding ZBF2/GBF1 bZIP protein) and ZBF3 (encoding ZBF3/CAM7 calmodulin protein), have been found to play crucial roles during these processes. ZBFs not only individually regulate the photomorphogenic growth but also coordinate among themselves and with other transcriptional networks to influence numerous aspects of plant growth and development. Significant progress has been made in recent years on the understanding of how ZBFs genetically and molecularly interact among themselves or with key regulatory components of light signaling pathways to control the developmental processes. In this review, we mostly focus on the current state of research of ZBFs in Arabidopsis seedling development.

Improving seed size, seed weight and seedling emergence in Camelina sativa by overexpressing the Atsob3-6 gene variant.

Seedling stand establishment is a critical factor affecting crop yield in low-precipitation agricultural regions. This is especially true for small seeded crops, such as Camelina (Camelina sativa) and canola (Brassica napus), that need to be planted shallow. Deeper planting would be desirable so that seeds can access soil moisture and bigger seeds could improve emergence and stand establishment by providing the energy necessary for seedling elongation. AHL (AT-Hook Containing, Nuclear Localized) genes play an important role in seedling growth and development. AHL proteins contain two structural units, the DNA-binding AT-hook motif and the Plant and Prokaryote Conserved (PPC) domain, required for protein-protein interactions. Our previous studies demonstrate that AtAHL29/SOB3 (Suppressor of phytochrome B-4 #3) regulates seedling development in Arabidopsis (Arabidopsis thaliana). Activation-tagged overexpression of AtSOB3 (Atsob3-D) represses the long-hypocotyl phenotype of an Arabidopsis phytochrome B mutant. In contrast, overexpression of the Atsob3-6 variant (Atsob3-6-OX), with a non-functional AT-hook, confers a long-hypocotyl phenotype. In this study, we demonstrate the role of Atsob3-D and Atsob3-6-OX in modulating seed size and hypocotyl length in the brassicas Arabidopsis and Camelina. In Arabidopsis, Atsob3-D reduces seed weight whereas Atsob3-6-OX increases seed weight and size when compared to the wild type. Similarly, Atsob3-6-OX transgenic Camelina seedlings are taller than the wild type, and produce larger and heavier seeds. These larger Atsob3-6-OX Camelina seeds also confer better emergence in deep-soil planting when compared to the wild type. Taken together, Atsob3-6-OX increases seed size, seed weight, seedling hypocotyl length and stand establishment in the oilseed crop Camelina.

Research on the Physio-Biochemical Mechanism of Non-Thermal Plasma-Regulated Seed Germination and Early Seedling Development in Arabidopsis.

Non-thermal plasma holds great potentials as an efficient, economical, and eco-friendly seed pretreatment method for improving the seed germination and seedling growth, but the mechanisms are still unclear. Therefore, a plant model organism Arabidopsis thaliana was used to investigate the physio-biochemical responses of seeds to non-thermal plasma at different treatment times by measuring the plant growth parameters, redox-related parameters, calcium (Ca2+) level and physicochemical modification of seed surface. The results showed that short-time plasma treatment (0.5, 1, and 3 min) promoted seed germination and seedling growth, whereas long-time plasma treatment (5 and 10 min) exhibited inhibitory effects. The level of superoxide anion (O2•−) and nitric oxide (NO) and the intensity of infrared absorption of the hydroxyl group were significantly higher in short-time plasma treated Arabidopsis seeds, and the level of hydrogen peroxide (H2O2) was remarkably increased in long-time plasma treated seeds, indicating that O2•−, ·OH, and NO induced by plasma may contribute to breaking seed dormancy and advancing seed germination in Arabidopsis, while plasma-induced H2O2 may inhibit the seed germination. The intensity of hydroxyl group and the contents of H2O2, malondialdehyde, and Ca2+ in Arabidopsis seedlings were obviously increased with the plasma treatment time. Catalase, superoxide dismutase, and peroxidase activities as well as proline level in short-time treated seedlings were apparently higher than in control. The etching effects of plasma on seed surface were dose-dependent, spanning from slight shrinkages to detached epidermis, which also significantly increased the oxidation degree of seed surface. Therefore, the improved activities of antioxidant systems, moderate ·OH, H2O2, and Ca2+ accumulation and seed surface modification induced by plasma all contribute to the enhanced seedling growth of Arabidopsis after short-time plasma treatment.

MYC2 regulates ARR16, a component of cytokinin signaling pathways, in Arabidopsis seedling development.

MYC2 is a basic helix‐loop‐helix transcription factor that acts as a repressor of blue light‐mediated photomorphogenic growth; however, it promotes lateral root formation. MYC2 also regulates different phytohormone‐signaling pathways in crucial manner. Arabidopsis response regulator 16 (ARR16) is a negative regulator of cytokinin signaling pathways. Here, we show that MYC2 directly binds to the E‐box of ARR16 minimal promoter and negatively regulates its expression in a cytokinin‐dependent manner. While ARR16 and MYC2 influence jasmonic acid and cytokinin signaling, the expression of ARR16 is regulated by cry1, GBF1, and HYH, the components of light signaling pathways. The transgenic studies show that the expression of ARR16 is regulated by MYC2 at various stages of development. The mutational studies reveal that ARR16 positively regulates the hypocotyl growth in blue light, and phenotypic analysis of atmyc2 arr16 double mutant further reveals that arr16 can suppress the short hypocotyl phenotype of atmyc2. Altogether, this work highlights MYC2‐mediated transcriptional repression of ARR16 in Arabidopsis seedling development.

Functional interrelation of MYC2 and HY5 plays an important role in Arabidopsis seedling development.

Arabidopsis MYC2 bHLH transcription factor plays a negative regulatory role in blue light (BL)-mediated seedling development. HY5 bZIP protein works as a positive regulator of multiple wavelengths of light and promotes photomorphogenesis. Both MYC2 and HY5, belonging to two different classes of transcription factors, are the integrators of multiple signaling pathways. However, the functional interrelations of these two transcription factors in seedling development remain unknown. Additionally, whereas HY5-mediated regulation of gene expression has been investigated in detail, the transcriptional regulation of HY5 itself is yet to be understood. Here, we show that HY5 and MYC2 work in an antagonistic manner in Arabidopsis seedling development. Our results reveal that HY5 expression is negatively regulated by MYC2 predominantly in BL, and at various stages of development. On the other hand, HY5 negatively regulates the expression of MYC2 at various wavelengths of light. In vitro and in vivo DNA-protein interaction studies suggest that MYC2 binds to the E-box cis-acting element of HY5 promoter. Collectively, this study demonstrates a coordinated regulation of MYC2 and HY5 in blue-light-mediated Arabidopsis seedling development.

COP1 regulates the stability of CAM7 to promote photomorphogenic growth.

The unique member of the calmodulin gene family, Calmodulin7 (CAM7), plays a crucial role as transcriptional regulator to promote Arabidopsis seedling development. CAM7 regulates the expression of HY5, which is intimately involved in the promotion of photomorphogenic growth and light‐regulated gene expression. COP1 ubiquitin ligase suppresses photomorphogenesis by degrading multiple photomorphogenesis promoting factors including HY5 in darkness. Genetic interaction studies, in this report, reveal that CAM7 and COP1 co‐ordinately work to promote photomorphogenic growth and light‐regulated gene expression at lower intensity of light. CAM7 physically interacts with COP1 in the nucleus. Further, in vivo study suggests that CAM7 and COP1 interaction is light intensity dependent. We have also shown that functional COP1 is required for optimum accumulation of CAM7 at lower fluences of light. Taken together, this study demonstrates the coordinated function of CAM7 and COP1 in Arabidopsis seedling development.

Requirement of ONE-HELIX PROTEIN 1 (OHP1) in early Arabidopsis seedling development and under high light intensity

ABSTRACTPlant ONE-HELIX PROTEINS (OHPs) are part of the light-harvesting complex superfamily whose members are involved in various processes related to sensing and capturing light as well as light protection. We recently showed the requirement of a functional OHP1-OHP2 heterodimer for efficient D1 synthesis. Interestingly, while the ohp1 knockout mutant showed a strong defect in accumulation of the photosystem II and is hardly viable, virus-induced gene silencing of OHP1 had no detectable impact on plant growth and performance under standard growth conditions. However, in vivo labeling assays with 35S-methionine indicate a reduced D1 synthesis rate. Here, we show that VIGS-OHP1 plants are more susceptible towards elevated light intensities than control plants. This underlines an obligatory function of OHP1 for light acclimation.

Analysis of a novel mutant allele of GSL8 reveals its key roles in cytokinesis and symplastic trafficking in Arabidopsis

BackgroundPlant cell walls are mainly composed of polysaccharides such as cellulose and callose. Callose exists at a very low level in the cell wall; however, it plays critical roles at different stages of plant development as well as in defence against unfavorable conditions. Callose is accumulated at the cell plate, at plasmodesmata and in male and female gametophytes. Despite the important roles of callose in plants, the mechanisms of its synthesis and regulatory properties are not well understood.ResultsCALLOSE SYNTHASE (CALS) genes, also known as GLUCAN SYNTHASE-LIKE (GSL), comprise a family of 12 members in Arabidopsis thaliana. Here, we describe a new allele of GSL8 (named essp8) that exhibits pleiotropic seedling defects. Reduction of callose deposition at the cell plates and plasmodesmata in essp8 leads to ectopic endomitosis and an increase in the size exclusion limit of plasmodesmata during early seedling development. Movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development, are dysregulated in the primary root of essp8. This observation provides evidence for a molecular mechanism explaining the gsl8 root phenotype. We demonstrated that GSL8 interacts with PLASMODESMATA-LOCALIZED PROTEIN 5, a β-1,3-glucanase, and GSL10. We propose that they all might be part of a putative callose synthase complex, allowing a concerted regulation of callose deposition at plasmodesmata.ConclusionAnalysis of a novel mutant allele of GSL8 reveals that GSL8 is a key player in early seedling development in Arabidopsis. GSL8 is required for maintaining the basic ploidy level and regulating the symplastic trafficking. Callose deposition at plasmodesmata is highly regulated and occurs through interaction of different components, likely to be incorporated into a callose biosynthesis complex. We are providing new evidence supporting an earlier hypothesis that GSL8 might have regulatory roles apart from its enzymatic function in plasmodesmata regulation.

Replication‐coupled histone H3.1 deposition determines nucleosome composition and heterochromatin dynamics during Arabidopsis seedling development

Developmental phase transitions are often characterized by changes in the chromatin landscape and heterochromatin reorganization. In Arabidopsis, clustering of repetitive heterochromatic loci into so-called chromocenters is an important determinant of chromosome organization in nuclear space. Here, we investigated the molecular mechanisms involved in chromocenter formation during the switch from a heterotrophic to a photosynthetically competent state during early seedling development. We characterized the spatial organization and chromatin features at centromeric and pericentromeric repeats and identified mutant contexts with impaired chromocenter formation. We find that clustering of repetitive DNA loci into chromocenters takes place in a precise temporal window and results in reinforced transcriptional repression. Although repetitive sequences are enriched in H3K9me2 and linker histone H1 before repeat clustering, chromocenter formation involves increasing enrichment in H3.1 as well as H2A.W histone variants, hallmarks of heterochromatin. These processes are severely affected in mutants impaired in replication-coupled histone assembly mediated by CHROMATIN ASSEMBLY FACTOR 1 (CAF-1). We further reveal that histone deposition by CAF-1 is required for efficient H3K9me2 enrichment at repetitive sequences during chromocenter formation. Taken together, we show that chromocenter assembly during post-germination development requires dynamic changes in nucleosome composition and histone post-translational modifications orchestrated by the replication-coupled H3.1 deposition machinery.