Anther development in Arabidopsis thaliana involves symplastic isolation and apoplastic gating of the tapetum-middle layer interface.

During flowering plant reproduction, anthers produce pollen grains, the development of which is supported by the tapetum, a nourishing maternal tissue that also contributes non-cell-autonomously to the pollen wall, the resistant external layer on the pollen surface. How the anther restricts movement of the tapetum-derived pollen wall components, while allowing metabolites such as sugars and amino acids to reach the developing pollen, remains unknown. Here, we show experimentally that in arabidopsis thaliana the tapetum and developing pollen are symplastically isolated from each other, and from other sporophytic tissues, from meiosis onwards. We show that the peritapetal strip, an apoplastic structure, separates the tapetum and the pollen grains from other anther cell layers and can prevent the apoplastic diffusion of fluorescent proteins, again from meiosis onwards. The formation and selective barrier functions of the peritapetal strip require two NADPH oxidases, RBOHE and RBOHC, which play a key role in pollen formation. Our results suggest that, together with symplastic isolation, gating of the apoplast around the tapetum may help generate metabolically distinct anther compartments.

Comparative analysis of wild-type accessions reveals novel determinants of Arabidopsis seed longevity.

Understanding the genetic factors involved in seed longevity is of paramount importance in agricultural and ecological contexts. The polygenic nature of this trait suggests that many of them remain undiscovered. Here, we exploited the contrasting seed longevity found amongst Arabidopsis thaliana accessions to further understand this phenomenon. Concentrations of glutathione were higher in longer-lived than shorter-lived accessions, supporting that redox poise plays a prominent role in seed longevity. However, high seed permeability, normally associated with shorter longevity, is also present in long-lived accessions. Dry seed transcriptome analysis indicated that the contribution to longevity of stored messenger RNA (mRNAs) is complex, including mainly accession-specific mechanisms. The detrimental effect on longevity caused by other factors may be counterbalanced by higher levels of specific mRNAs stored in dry seeds, for instance those of heat-shock proteins. Indeed, loss-of-function mutant analysis demonstrated that heat-shock factors HSF1A and 1B contributed to longevity. Furthermore, mutants of the stress-granule zinc-finger protein TZF9 or the spliceosome subunits MOS4 or MAC3A/MAC3B, extended seed longevity, positioning RNA as a novel player in the regulation of seed viability. mRNAs of proteins with putative relevance to longevity were also abundant in shorter-lived accessions, reinforcing the idea that resistance to ageing is determined by multiple factors.

Seed coat lignification level is crucial in Capsicum spp seed longevity.

Capsicum (pepper) is known for its poor seed germination, particularly seed longevity is usually much shorter than other Solanaceae. However, the molecular mechanisms involved are mostly unknown in these species. The present study examines the differences in seed longevity among Capsicum species and varietal types. Feral or less domesticated species, such as Capsicum chinense and particularly Capsicum frutescens, showed higher germination rates than the more domesticated Capsicum annuum after accelerated seed aging treatments. In addition, variability was detected in the expression of genes involved in the response to seed deterioration. The differences observed in ASPG1 expression led us to study the seed protein profile in dry and germinating seeds. Seed storage protein mobilization during germination was faster in seed aging-resistant genotypes. Similarly, the transcriptional change observed for the orthologous gene of the trans-species regulator AtHB25 prompted us to study the structure and molecular components of the seed coat in peppers. All the Capsicum pepper accessions analyzed presented very lignified testa and we observed a positive correlation between the amount of lignin and seed viability. Our results provide essential information to explain the poor germination observed in pepper seeds and provide an experimental framework for future improvements in this important character.

Apoplastic lipid barriers regulated by conserved homeobox transcription factors extend seed longevity in multiple plant species.

Cutin and suberin are lipid polyesters deposited in specific apoplastic compartments. Their fundamental roles in plant biology include controlling the movement of gases, water and solutes, and conferring pathogen resistance. Both cutin and suberin have been shown to be present in the Arabidopsis seed coat where they regulate seed dormancy and longevity. In this study, we use accelerated and natural ageing seed assays, glutathione redox potential measures, optical and transmission electron microscopy and gas chromatography-mass spectrometry to demonstrate that increasing the accumulation of lipid polyesters in the seed coat is the mechanism by which the AtHB25 transcription factor regulates seed permeability and longevity. Chromatin immunoprecipitation during seed maturation revealed that the lipid polyester biosynthetic gene long-chain acyl-CoA synthetase 2 (LACS2) is a direct AtHB25 binding target. Gene transfer of this transcription factor to wheat and tomato demonstrated the importance of apoplastic lipid polyesters for the maintenance of seed viability. Our work establishes AtHB25 as a trans-species regulator of seed longevity and has identified the deposition of apoplastic lipid barriers as a key parameter to improve seed longevity in multiple plant species.

Identification of novel seed longevity genes related to oxidative stress and seed coat by genome-wide association studies and reverse genetics.

Seed longevity is a polygenic trait of relevance for agriculture and for understanding the effect of environment on the ageing of biological systems. In order to identify novel longevity genes, we have phenotyped the natural variation of 270 ecotypes of the model plant, Arabidopsis thaliana, for natural ageing and for three accelerated ageing methods. Genome-wide analysis, using publicly available single-nucleotide polymorphisms (SNPs) data sets, identified multiple genomic regions associated with variation in seed longevity. Reverse genetics of 20 candidate genes in Columbia ecotype resulted in seven genes positive for seed longevity (PSAD1, SSLEA, SSTPR, DHAR1, CYP86A8, MYB47 and SPCH) and five negative ones (RBOHD, RBOHE, RBOHF, KNAT7 and SEP3). In this uniform genetic background, natural and accelerated ageing methods provided similar results for seed-longevity in knock-out mutants. The NADPH oxidases (RBOHs), the dehydroascorbate reductase (DHAR1) and the photosystem I subunit (PSAD1) highlight the important role of oxidative stress on seed ageing. The cytochrome P-450 hydroxylase, CYP86A8, and the transcription factors, MYB47, KNAT7 and SEP3, support the protecting role of the seed coat during seed ageing.

PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis.

Permeability is a crucial trait that affects seed longevity and is regulated by different polymers including proanthocyanidins, suberin, cutin and lignin located in the seed coat. By testing mutants in suberin transport and biosynthesis, we demonstrate the importance of this biopolymer to cope with seed deterioration. Transcriptomic analysis of cog1-2D, a gain-of-function mutant with increased seed longevity, revealed the upregulation of several peroxidase genes. Reverse genetics analysing seed longevity uncovered redundancy within the seed coat peroxidase gene family; however, after controlled deterioration treatment, seeds from the prx2 prx25 double and prx2 prx25 prx71 triple mutant plants presented lower germination than wild-type plants. Transmission electron microscopy analysis of the seed coat of these mutants showed a thinner palisade layer, but no changes were observed in proanthocyanidin accumulation or in the cuticle layer. Spectrophotometric quantification of acetyl bromide-soluble lignin components indicated changes in the amount of total polyphenolics derived from suberin and/or lignin in the mutant seeds. Finally, the increased seed coat permeability to tetrazolium salts observed in the prx2 prx25 and prx2 prx25 prx71 mutant lines suggested that the lower permeability of the seed coats caused by altered polyphenolics is likely to be the main reason explaining their reduced seed longevity.

The plant cuticle.

The plant cuticle is one of the key innovations that allowed plants to colonize terrestrial ecosystems. By limiting molecular diffusion, the cuticle provides an interface that ensures controlled interactions between plant surfaces and their environments. It confers diverse and sometimes astonishing properties upon plant surfaces at both the molecular level (from water and nutrient exchange capacities to almost complete impermeability), to the macroscopic level (from water repellence to iridescence). It takes the form of a continuous modification of the outer cell wall of the plant epidermis from early in plant development (surrounding the epidermis of the developing plant embryo) and is actively maintained and modified throughout the growth and development of most plant aerial organs - including non-woody stems, flowers, leaves, and even the root cap of emerging primary and lateral roots. The cuticle was first identified as a distinct structure in the early 19th century, and has since been the focus of intense research that, while revealing the fundamental role of the cuticle in the life of terrestrial plants, has also highlighted many unresolved mysteries regarding cuticle biogenesis and structure.

Endosperm Persistence in Arabidopsis Results in Seed Coat Fractures and Loss of Seed Longevity.

Seeds are specialized plant organs that carry, nurture, and protect plant offspring. Developmental coordination between the three genetically distinct seed tissues (the embryo, endosperm, and seed coat) is crucial for seed viability. In this study, we explore the relationship between the TFs AtHB25 and ICE1. Previous results identified ICE1 as a target gene of AtHB25. In seeds, a lack of ICE1 (ice1-2) suppresses the enhanced seed longevity and impermeability of the overexpressing mutant athb25-1D, but surprisingly, seed coat lipid polyester deposition is not affected, as shown by the double-mutant athb25-1D ice1-2 seeds. zou-4, another mutant lacking the transcriptional program for proper endosperm maturation and for which the endosperm persists, also presents a high sensitivity to seed aging. Analysis of gso1, gso2, and tws1-4 mutants revealed that a loss of embryo cuticle integrity does not underlie the seed-aging sensitivity of ice1-2 and zou-4. However, scanning electron microscopy revealed the presence of multiple fractures in the seed coats of the ice1 and zou mutants. Thus, this study highlights the importance of both seed coat composition and integrity in ensuring longevity and demonstrates that these parameters depend on multiple factors.

Transcription Factor DOF4.1 Regulates Seed Longevity in Arabidopsis via Seed Permeability and Modulation of Seed Storage Protein Accumulation.

Seed longevity is modulated by multiple genetic factors in Arabidopsis thaliana. A previous genome-wide association study using the Elevated Partial Pressure of Oxygen (EPPO) aging assay pinpointed a genetic locus associated with this trait. Reverse genetics identified the transcription factor DOF4.1 as a novel seed longevity factor. dof4.1 loss-of-function plants generate seeds exhibiting higher germination after accelerated aging assays. DOF4.1 is expressed during seed development and RNAseq data show several putative factors that could contribute to the dof4.1 seed longevity phenotype. dof4.1 has reduced seed permeability and a higher levels of seed storage proteins mRNAs (cruciferins and napins) in developing seeds, as compared to wild-type seeds. It has been reported that mutant lines defective in cruciferins or napins present reduced seed longevity. The improved longevity of dof4.1 is totally lost in the quadruple mutant dof4.1 cra crb crc, but not in a dof4.1 line depleted of napins, suggesting a prominent role for cruciferins in this process. Moreover, a negative regulation of DOF4.1 expression by the transcription factor DOF1.8 is suggested by co-inoculation assays in Nicotiana benthamiana. Indeed, DOF1.8 expression anticorrelates with that of DOF4.1 during seed development. In summary, modulation of DOF4.1 levels during seed development contributes to regulate seed longevity.