Embryo Development In Arabidopsis Research Articles

PLETHORA transcription factors promote early embryo development through induction of meristematic potential.

Plants are dependent on divisions of stem cells to establish cell lineages required for growth. During embryogenesis, early division products are considered to be stem cells, whereas during post-embryonic development, stem cells are present in meristems at the root and shoot apex. PLETHORA/AINTEGUMENTA-LIKE (PLT/AIL) transcription factors are regulators of post-embryonic meristem function and are required to maintain stem cell pools. Despite the parallels between embryonic and post-embryonic stem cells, the role of PLTs during early embryogenesis has not been thoroughly investigated. Here, we demonstrate that the PLT regulome in the zygote, and apical and basal cells is in strong congruence with that of post-embryonic meristematic cells. We reveal that out of all six PLTs, only PLT2 and PLT4/BABY BOOM (BBM) are expressed in the zygote, and that these two factors are essential for progression of embryogenesis beyond the zygote stage and first divisions. Finally, we show that other PLTs can rescue plt2 bbm defects when expressed from the PLT2 and BBM promoters, establishing upstream regulation as a key factor in early embryogenesis. Our data indicate that generic PLT factors facilitate early embryo development in Arabidopsis by induction of meristematic potential.

Two MADS-box proteins, AGL9 and AGL15, recruit the FIS-PRC2 complex to trigger the phase transition from endosperm proliferation to embryo development in Arabidopsis

Spatiotemporal regulation of gene expression by polycomb repressive complex 2 (PRC2) is critical for animal and plant development. The Arabidopsis fertilization independent seed (FIS)-PRC2 complex functions specifically during plant reproduction from gametogenesis to seed development. After a double fertilization event, triploid endosperm proliferates early, followed by the growth of a diploid embryo, which replaces the endosperm in Arabidopsis and many dicots. Key genes critical for endosperm proliferation such as IKU2 and MINI3 are activated after fertilization. Here we report that two MADS-box AGAMOUS-LIKE (AGL) proteins associate with the key endosperm proliferation loci and recruit the FIS-PRC2 repressive complex at 4–5 days after pollination (DAP). Interestingly, AGL9 and AGL15 only accumulate toward the end of endosperm proliferation at 4–5 DAP and promote the deposition of H3K27me3 marks at key endosperm proliferation loci. Disruption of AGL9 and AGL15 or overexpression of AGL9 or AGL15 significantly influence endosperm proliferation and cellularization. Genome-wide analysis with cleavage Under Targets and tagmentation (CUT&Tag) sequencing and RNA sequencing revealed the landscape of endosperm H3K27me3 marks and gene expression profiles in Col-0 and agl9 agl15. CUT&Tag qPCR also demonstrated the occupancy of the two MADS-box proteins and FIS-PRC2 on a few representative target loci. Our studies suggest that MADS-box proteins could potentially recruit PRC2 to regulate many other developmental processes in plants or even in fungi and animals.

Arabidopsis ribosomal RNA processing meerling mutants exhibit suspensor-derived polyembryony due to direct reprogramming of the suspensor.

Embryo development in Arabidopsis (Arabidopsis thaliana) starts off with an asymmetric division of the zygote to generate the precursors of the embryo proper and the supporting extraembryonic suspensor. The suspensor degenerates as the development of the embryo proper proceeds beyond the heart stage. Until the globular stage, the suspensor maintains embryonic potential and can form embryos in the absence of the developing embryo proper. We report a mutant called meerling-1 (mrl-1), which shows a high penetrance of suspensor-derived polyembryony due to delayed development of the embryo proper. Eventually, embryos from both apical and suspensor lineages successfully develop into normal plants and complete their life cycle. We identified the causal mutation as a genomic rearrangement altering the promoter of the Arabidopsis U3 SMALL NUCLEOLAR RNA-ASSOCIATED PROTEIN 18 (UTP18) homolog that encodes a nucleolar-localized WD40-repeat protein involved in processing 18S preribosomal RNA. Accordingly, root-specific knockout of UTP18 caused growth arrest and accumulation of unprocessed 18S pre-rRNA. We generated the mrl-2 loss-of-function mutant and observed asynchronous megagametophyte development causing embryo sac abortion. Together, our results indicate that promoter rearrangement decreased UTP18 protein abundance during early stage embryo proper development, triggering suspensor-derived embryogenesis. Our data support the existence of noncell autonomous signaling from the embryo proper to prevent direct reprogramming of the suspensor toward embryonic fate.

Endogenous auxin maintains embryonic cell identity and promotes somatic embryo development in Arabidopsis.

Somatic embryogenesis (SE), or embryo development from invitro cultured vegetative explants, can be induced in Arabidopsis by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or by overexpression of specific transcription factors, such as AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15). Here, we explored the role of endogenous auxin [indole-3-acetic acid (IAA)] during 2,4-D and AHL15-induced SE. Using the pWOX2:NLS-YFP reporter, we identified three distinct developmental stages for 2,4-D and AHL15-induced SE in Arabidopsis, with these being (i) acquisition of embryo identity; (ii) formation of pro-embryos; and (iii) somatic embryo patterning and development. The acquisition of embryo identity coincided with enhanced expression of the indole-3-pyruvic acid auxin biosynthesis YUCCA genes, resulting in an enhanced pDR5:GFP-reported auxin response in the embryo-forming tissues. Chemical inhibition of the indole-3-pyruvic acid pathway did not affect the acquisition of embryo identity, but significantly reduced or completely inhibited the formation of pro-embryos. Co-application of IAA with auxin biosynthesis inhibitors in the AHL15-induced SE system rescued differentiated somatic embryo formation, confirming that increased IAA levels are important during the last two stages of SE. Our analyses also showed that polar auxin transport, with AUXIN/LIKE-AUX influx and PIN-FORMED1 efflux carriers as important drivers, is required for the transition of embryonic cells to proembryos and, later, for correct cell fate specification and differentiation. Taken together, our results indicate that endogenous IAA biosynthesis and its polar transport are not required for the acquisition of embryo identity, but rather to maintain embryonic cell identity and for the formation of multicellular proembryos and their development into histodifferentiated embryos.

The dynamic proteome in Arabidopsis thaliana early embryogenesis.

The morphology of the flowering plant is established during early embryogenesis. In recent years, many studies have focused on transcriptional profiling in plant embryogenesis, but the dynamic landscape of the Arabidopsis thaliana proteome remains elusive. In this study, Arabidopsis embryos at 2/4-cell, 8-cell, 16-cell, 32-cell, globular and heart stages were collected for nanoproteomic analysis. In total, 5386 proteins were identified. Of these, 1051 proteins were universally identified in all developmental stages and a range of 27 to 2154 proteins was found to be stage specific. These proteins could be grouped into eight clusters according to their expression levels. Gene Ontology enrichment analysis showed that genes involved in ribosome biogenesis and auxin-activated signalling were enriched during early embryogenesis, indicating that active translation and auxin signalling are important events in Arabidopsis embryo development. Combining RNA-sequencing data with the proteomics analysis, the correlation between mRNA and protein was evaluated. An overall positive correlation was found between mRNA and protein. This work provides a comprehensive landscape of the Arabidopsis proteome in early embryogenesis. Some important proteins/transcription factors identified through network analysis may serve as potential targets for future investigation.

EIF4E1 Regulates Arabidopsis Embryo Development and Root Growth by Interacting With RopGEF7.

Eukaryotic translation initiation factor 4E1 (eIF4E1) is required for the initiation of protein synthesis. The biological function of eIF4E1 in plant–potyvirus interactions has been extensively studied. However, the role of eIF4E1 in Arabidopsis development remains unclear. In this study, we show that eIF4E1 is highly expressed in the embryo and root apical meristem. In addition, eIF4E1 expression is induced by auxin. eIF4E1 mutants show embryonic cell division defects and short primary roots, a result of reduced cell divisions. Furthermore, our results show that mutation in eIF4E1 severely reduces the accumulation of PIN-FORMED (PIN) proteins and decreases auxin-responsive gene expression at the root tip. Yeast two-hybrid assays identified that eIF4E1 interacts with an RAC/ROP GTPase activator, RopGEF7, which has been previously reported to be involved in the maintenance of the root apical meristem. The interaction between eIF4E1 and RopGEF7 is confirmed by protein pull-down and bimolecular fluorescent complementation assays in plant cells. Taken together, our results demonstrated that eIF4E1 is important for auxin-regulated embryo development and root growth. The eIF4E1–RopGEF7 interaction suggests that eIF4E1 may act through ROP signaling to regulate auxin transport, thus regulating auxin-dependent patterning.

MISF2 Encodes an Essential Mitochondrial Splicing Cofactor Required for nad2 mRNA Processing and Embryo Development in Arabidopsis thaliana.

Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players in plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1, and thus respiratory complex I biogenesis, are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot–dicot divergence ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.

Glucuronidation of type II arabinogalactan polysaccharides function in sexual reproduction of Arabidopsis.

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.

Arabidopsis AtPRP17 functions in embryo development by regulating embryonic patterning.

Arabidopsis AtPRP17, a homolog of yeast splicing factor gene PRP17, is expressed in siliques and embryos and functions in embryo development via regulating embryonic patterning. Yeast splicing factor PRP17/CDC40 is essential for cell growth through involvement in cell cycle regulation. Arabidopsis genome encodes a homolog of PRP17, AtPRP17; however, its function in Arabidopsis development is unknown. This study showed that AtPRP17 was highly expressed in siliques and embryos, and the protein was localized in the nucleus. The loss-of-function mutation of AtPRP17 led to shrunken seeds in Arabidopsis mature siliques. Further analysis revealed that the defective mature seeds of the mutant resulted from abnormal embryos with shriveled cotyledons, unequal cotyledons, swollen and shortened hypocotyls, or shortened radicles. During embryogenesis, mutant embryos showed delayed development and defective patterning of the apical and base domains, such as inhibited cotyledons and disorganized quiescent center cells and columella. Our results suggested that AtPRP17 functions in Arabidopsis embryo development via regulating embryonic patterning.

Plastid-Localized EMB2726 Is Involved in Chloroplast Biogenesis and Early Embryo Development in Arabidopsis.

Embryogenesis is a critical developmental process that establishes the body organization of higher plants. During this process, the biogenesis of chloroplasts from proplastids is essential. A failure in chloroplast development during embryogenesis can cause morphologically abnormal embryos or embryonic lethality. In this study, we isolated a T-DNA insertion mutant of the Arabidopsis gene EMBRYO DEFECTIVE 2726 (EMB2726). Heterozygous emb2726 seedlings produced about 25% albino seeds with embryos that displayed defects at the 32-cell stage and that arrested development at the late globular stage. EMB2726 protein was localized in chloroplasts and was expressed at all stages of development, such as embryogenesis. Moreover, the two translation elongation factor Ts domains within the protein were critical for its function. Transmission electron microscopy revealed that the cells in emb2726 embryos contained undifferentiated proplastids and that the expression of plastid genome-encoded photosynthesis-related genes was dramatically reduced. Expression studies of DR5:GFP, pDRN:DRN-GFP, and pPIN1:PIN1-GFP reporter lines indicated normal auxin biosynthesis but altered polar auxin transport. The expression of pSHR:SHR-GFP and pSCR:SCR-GFP confirmed that procambium and ground tissue precursors were lacking in emb2726 embryos. The results suggest that EMB2726 plays a critical role during Arabidopsis embryogenesis by affecting chloroplast development, possibly by affecting the translation process in plastids.

RUS6, a DUF647-containing protein, is essential for early embryonic development in Arabidopsis thaliana

BackgroundThe Arabidopsis RUS (ROOT UV-B SENSITIVE) gene family contains six members, each of which encodes a protein containing a DUF647 (domain of unknown function 647) that is commonly found in eukaryotes. Previous studies have demonstrated that RUS1 and RUS2 play critical roles in early seedling development. All six RUS genes are expressed throughout the plant, but little is known about the functional roles of RUS3, RUS4, RUS5 and RUS6.ResultsWe used a reverse-genetic approach to identify knockout mutants for RUS3, RUS4, RUS5 and RUS6. Each mutant was confirmed by direct DNA sequencing and genetic segregation analysis. No visible phenotypic differences were observed in rus3, rus4, or rus5 knockout mutants under standard growth conditions, but rus6 knockout mutants displayed a strong embryo-lethal phenotype. Two independent knockout lines for RUS6 were characterized. The rus6 mutations could only be maintained through a heterozygote, because rus6 homozygous mutants did not survive. Closer examinations of homozygous rus6 embryos from rus6/ + parent plants revealed that RUS6 is required for early embryo development. Loss of RUS6 resulted in embryo lethality, specifically at the mid-globular stage. The embryo-lethality phenotype was complemented by a RUS6::RUS6-GFP transgene, and GFP signal was detected throughout the embryo. Histological analyses with the β-glucuronidase reporter gene driven by the RUS6 promoter showed tissue- and development-specific expression of RUS6, which was highest in floral tissues.ConclusionOur data revealed that RUS6 is essential for early embryo development in Arabidopsis, and that the RUS gene family functions in multiple stages of plant development.

Conserved, divergent and heterochronic gene expression during Brachypodium and Arabidopsis embryo development

Key messageDevelopmental and transcriptomic analysis of Brachypodium embryogenesis and comparison with Arabidopsis identifies conserved and divergent phases of embryogenesis and reveals widespread heterochrony of developmental gene expression.Embryogenesis, transforming the zygote into the mature embryo, represents a fundamental process for all flowering plants. Current knowledge of cell specification and differentiation during plant embryogenesis is largely based on studies of the dicot model plant Arabidopsis thaliana. However, the major crops are monocots and the transcriptional programs associated with the differentiation processes during embryogenesis in this clade were largely unknown. Here, we combined analysis of cell division patterns with development of a temporal transcriptomic resource during embryogenesis of the monocot model plant Brachypodium distachyon. We found that early divisions of the Brachypodium embryo were highly regular, while later stages were marked by less stereotypic patterns. Comparative transcriptomic analysis between Brachypodium and Arabidopsis revealed that early and late embryogenesis shared a common transcriptional program, whereas mid-embryogenesis was divergent between species. Analysis of orthology groups revealed widespread heterochronic expression of potential developmental regulators between the species. Interestingly, Brachypodium genes tend to be expressed at earlier stages than Arabidopsis counterparts, which suggests that embryo patterning may occur early during Brachypodium embryogenesis. Detailed investigation of auxin-related genes shows that the capacity to synthesize, transport and respond to auxin is established early in the embryo. However, while early PIN1 polarity could be confirmed, it is unclear if an active response is mounted. This study presents a resource for studying Brachypodium and grass embryogenesis and shows that divergent angiosperms share a conserved genetic program that is marked by heterochronic gene expression.

DEMETER-mediated DNA Demethylation in Gamete Companion Cells and the Endosperm, and its Possible Role in Embryo Development in Arabidopsis

Seed development begins upon double fertilization, producing the embryo and endosperm, which are genetically identical, except for their ploidy level. DEMETER (DME), a member of the DNA glycosylase family, functions as a DNA demethylase via the base excision repair pathway. DME is specifically expressed prior to fertilization in two gamete companion cells, central cell of the female gametophyte and vegetative cell of the male gametophyte, but not in the heritable gamete cells or embryo. Mutations in the DME gene cause hypermethylation in the endosperm, leading to endosperm overproliferation and seed abortion after fertilization. DME-mediated DNA demethylation preferentially targets euchromatic transposable elements (TEs), resulting in TE activation and initiation of de novo methylation through RNA-directed DNA methylation, and provides FERTILIZATION-INDEPENDENT SEED 2 (FIS2)-Polycomb Repressive Complex 2-binding sites, resulting in histone modifications and genomic imprinting during reproduction. The global demethylation of TEs in gamete companion cells and active de novo methylation in the embryo suggest a new role of sexual companion cells in reinforcing the genome integrity of the heritable tissue. In this review, we provide an overview of demethylation in sexual companion cells and the endosperm, and discuss its evolutionary effect on the heritable gamete cells and embryo.

AGC protein kinase AGC1-4 mediates seed size in Arabidopsis.

AGC1-4 kinase plays a crucial role in the regulation of seeds by mediating cell proliferation and embryo development in Arabidopsis. Seed size is a crucial factor to influence final seed yield in plants. However, the molecular mechanisms that set final seed size still need to be investigated. Here, we identified a novel AGC protein kinase AGC1-4, which encodes a serine-threonine kinase, belongs to the AGC VIIIa subfamily. The seeds of agc1-4 mutant were significantly larger than that in the wild type. Overexpression of the AGC1-4 gene reduced seed size. Regulation of AGC1-4 seed size is dependent on embryonic cell number. To further determine AGC1-4 functions in seed size, we analyzed AGC1-4 phosphoproteins using label-free quantitative phosphoproteomics coupled to the transcriptome of agc1-4 using RNA sequencing (RNA-seq). The RNA-seq analysis showed 1611 differentially expressed genes (DEGs), which cover a wide range of functions, such as cell cycle and embryo development. The 262 unique phosphoproteins were detected by phosphoproteomics analysis. The differentially phosphorylated proteins were involved in cell cycle and post-embryo development. Overlay of the RNA-seq and phosphoproteomics results demonstrated AGC1-4 as an important factor that influences seed size by mediating cell proliferation and embryo development. The results in this study provide novel data on the serine-threonine kinase AGC1-4 mediating seed size in Arabidopsis.

The Arabidopsis TRM61/TRM6 complex is a bona fide tRNA N1-methyladenosine methyltransferase.

tRNA molecules, which contain the most abundant post-transcriptional modifications, are crucial for proper gene expression and protein biosynthesis. Methylation at N1 of adenosine 58 (A58) is critical for maintaining the stability of initiator methionyl-tRNA (tRNAiMet) in bacterial, archaeal, and eukaryotic tRNAs. However, although research has been conducted in yeast and mammals, it remains unclear how A58 in plant tRNAs is modified and involved in development. In this study, we identify the nucleus-localized complex AtTRM61/AtTRM6 in Arabidopsis as tRNA m1A58 methyltransferase. Deficiency or a lack of either AtTRM61 or AtTRM6 leads to embryo arrest and seed abortion. The tRNA m1A level decreases in conditionally complemented Attrm61/LEC1pro::AtTRM61 plants and this is accompanied by reduced levels of tRNAiMet, indicating the importance of the tRNA m1A modification for tRNAiMet stability. Taken together, our results demonstrate that tRNA m1A58 modification is necessary for tRNAiMet stability and is required for embryo development in Arabidopsis.

Combined GWAS and QTL analysis for dissecting the genetic architecture of kernel test weight in maize.

Kernel weight in a unit volume is referred to as kernel test weight (KTW) that directly reflects maize (Zea mays L.) grain quality. In this study, an inter-mated B73 × Mo17 (IBM) Syn10 doubled haploid (DH) population and an association panel were used to identify loci responsible for KTW of maize across multiple environments. A total of 18 significant KTW-related single-nucleotide polymorphisms (SNPs) were identified using genome-wide association study (GWAS); they were closely linked to 12 candidate genes. In the IBM Syn10 DH population, linkage analysis detected 19 common quantitative trait loci (QTL), five of which were repeatedly detected among multiple environments. Several verified genes that regulate maize seed development were found in the confidence intervals of the mapped QTL and the LD regions of GWAS, such as ZmYUC1, BAP2, ZmTCRR-1, dek36 and ZmSWEET4c. Combined QTL mapping and GWAS identified one significant SNP that was co-identified in the both populations. Based on the co-localized SNP across the both populations, 17 candidate genes were identified. Of them, Zm00001d044075, Zm00001d044086, and Zm00001d044081 were further identified by candidate gene association study for KTW. Zm00001d044081 encodes homeobox-leucine zipper protein ATHB-4, which has been demonstrated to control apical embryo development in Arabidopsis. Our findings provided insights into the mechanism underlying maize KTW and contributed to the application of molecular-assisted selection of high KTW breeding in maize.

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana.

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.

Two tonoplast proton pumps function in Arabidopsis embryo development.

Two types of tonoplast proton pumps, H+ -pyrophosphatase (V-PPase) and the H+ -ATPase (V-ATPase), establish the proton gradient that powers molecular traffic across the tonoplast thereby facilitating turgor regulation and nutrient homeostasis. However, how proton pumps regulate development remains unclear. In this study, we investigated the function of two types of proton pumps in Arabidopsis embryo development and pattern formation. While disruption of either V-PPase or V-ATPase had no obvious effect on plant embryo development, knocking out both resulted in severe defects in embryo pattern formation from the early stage. While the first division in wild-type zygote was asymmetrical, a nearly symmetrical division occurred in the mutant, followed by abnormal pattern formation at all stages of embryo development. The embryonic defects were accompanied by dramatic differences in vacuole morphology and distribution, as well as disturbed localisation of PIN1. The development of mutant cotyledons and root, and the auxin response of mutant seedlings supported the hypothesis that mutants lacking tonoplast proton pumps were defective in auxin transport and distribution. Taking together, we proposed that two tonoplast proton pumps are required for vacuole morphology and PIN1 localisation, thereby controlling vacuole and auxin-related developmental processes in Arabidopsis embryos and seedlings.

AtNSE1 and AtNSE3 are required for embryo pattern formation and maintenance of cell viability during Arabidopsis embryogenesis.

Embryogenesis is an essential process during seed development in higher plants. It has previously been shown that mutation of the Arabidopsis non-SMC element genes AtNSE1 or AtNSE3 leads to early embryo abortion, and their proteins can interact with each other directly. However, the crucial regions of these proteins in this interaction and how the proteins are cytologically involved in Arabidopsis embryo development are unknown. In this study, we found that the C-terminal including the Ring-like motif of AtNSE1 can interact with the N-terminal of AtNSE3, and only the Ring-like motif is essential for binding with three α motifs of AtNSE2 (homologous to AtMMS21). Using genetic assays and by analysing molecular markers of cell fate decisions (STM, WOX5, and WOX8) in mutant nse1 and nse3 embryos, we found that AtNSE1 and AtNSE3 work non-redundantly in early embryo development, and that differentiation of the apical meristem and the hypophysis fails in the mutants, which have disrupted auxin transportation and responses. However, the upper cells of the suspensor in the mutants seem to have proper embryo cell identity. Cytological examination showed that cell death occurred from the early embryo stage, and that vacuolar programmed cell death and necrosis in the nse1 and nse3 mutant embryos led to ovule abortion. Thus, AtNSE1 and AtNSE3 are essential for maintaining cell viability and growth during early embryogenesis. Our results improve our understanding of the functions of SMC5/6 complex in early embryogenesis in Arabidopsis.

The plastidial pentose phosphate pathway is essential for postglobular embryo development in Arabidopsis

Large numbers of genes essential for embryogenesis in Arabidopsis encode enzymes of plastidial metabolism. Disruption of many of these genes results in embryo arrest at the globular stage of development. However, the cause of lethality is obscure. We examined the role of the plastidial oxidative pentose phosphate pathway (OPPP) in embryo development. In nonphotosynthetic plastids the OPPP produces reductant and metabolic intermediates for central biosynthetic processes. Embryos with defects in various steps in the oxidative part of the OPPP had cell division defects and arrested at the globular stage, revealing an absolute requirement for the production via these steps of ribulose-5-phosphate. In the nonoxidative part of the OPPP, ribulose-5-phosphate is converted to ribose-5-phosphate (R5P)-required for purine nucleotide and histidine synthesis-and subsequently to erythrose-4-phosphate, which is required for synthesis of aromatic amino acids. We show that embryo development through the globular stage specifically requires synthesis of R5P rather than erythrose-4-phosphate. Either a failure to convert ribulose-5-phosphate to R5P or a block in purine nucleotide biosynthesis beyond R5P perturbs normal patterning of the embryo, disrupts endosperm development, and causes early developmental arrest. We suggest that seed abortion in mutants unable to synthesize R5P via the oxidative part of the OPPP stems from a lack of substrate for synthesis of purine nucleotides, and hence nucleic acids. Our results show that the plastidial OPPP is essential for normal developmental progression as well as for growth in the embryo.