Stepwise changes in flavonoids in spores/pollen contributed to terrestrial adaptation of plants.

Protecting haploid pollen and spores against UV-B light and high temperature, 2 major stresses inherent to the terrestrial environment, is critical for plant reproduction and dispersal. Here, we show flavonoids play an indispensable role in this process. First, we identified the flavanone naringenin, which serves to defend against UV-B damage, in the sporopollenin wall of all vascular plants tested. Second, we found that flavonols are present in the spore/pollen protoplasm of all euphyllophyte plants tested and that these flavonols scavenge reactive oxygen species to protect against environmental stresses, particularly heat. Genetic and biochemical analyses showed that these flavonoids are sequentially synthesized in both the tapetum and microspores during pollen ontogeny in Arabidopsis (Arabidopsis thaliana). We show that stepwise increases in the complexity of flavonoids in spores/pollen during plant evolution mirror their progressive adaptation to terrestrial environments. The close relationship between flavonoid complexity and phylogeny and its strong association with pollen survival phenotypes suggest that flavonoids played a central role in the progression of plants from aquatic environments into progressively dry land habitats.

The regulatory mechanism of rapid lignification for timely anther dehiscence.

Anther dehiscence is a crucial event in plant reproduction, tightly regulated and dependent on the lignification of the anther endothecium. In this study, we investigated the rapid lignification process that ensures timely anther dehiscence in Arabidopsis. Our findings reveal that endothecium lignification can be divided into two distinct phases. During Phase I, lignin precursors are synthesized without polymerization, while Phase II involves simultaneous synthesis of lignin precursors and polymerization. The transcription factors MYB26, NST1/2, and ARF17 specifically regulate the pathway responsible for the synthesis and polymerization of lignin monomers in Phase II. MYB26-NST1/2 is the key regulatory pathway responsible for endothecium lignification, while ARF17 facilitates this process by interacting with MYB26. Interestingly, our results demonstrate that the lignification of the endothecium, which occurs within approximately 26 h, is much faster than that of the vascular tissue. These findings provide valuable insights into the regulation mechanism of rapid lignification in the endothecium, which enables timely anther dehiscence and successful pollen release during plant reproduction.

Efficient production of GlcNAc in an aqueous-organic system with a Chitinolyticbacter meiyuanensis SYBC-H1 mutant.

OBJECTIVES: Shellfish waste is a primary source for making N-acetyl-D-glucosamine. Thus, establishing a high-efficiency and low-cost bioconversion method to produce N-acetyl-D-glucosamine directly from shellfish waste was promising. RESULTS: A mutant C81 was obtained from Chitinolyticbacter meiyuanensis SYBC-H1 via 60Co-γ irradiation. This mutant C81 showed the highest chitinase activity of 9.8 U/mL that was 85% higher than the parent strain. The mutant C81 exhibted improved antioxidant activities, including total antioxidant capacity, superoxide radical ability, and hydroxyl radical scavenging ability, compared to that of the parent strain. Four out of nine organic solvents increased the chitinase activity by 1.9%, 6.8%, 11.7%, and 15.8%, corresponding to methylbenzene, n-heptane, petroleum ether, and n-hexane, respectively. The biphase system composed of aqueous and hexane presented a five-fold reduction of cell viability compared to the control. Using a continuous fermentation bioconversion process, 4.2 g/L GlcNAc was produced from crayfish shell powder with a yield of 80% of the chitin content. CONCLUSIONS: This study demonstrated that the mutant C81 is suitable for converting crayfish shell powder into GlcNAc in an aqueous-organic system.

AUXIN RESPONSE FACTOR17 Directly Regulates MYB108 for Anther Dehiscence.

The timely release of mature pollen following anther dehiscence is essential for reproduction in flowering plants. AUXIN RESPONSE FACTOR17 (ARF17) plays a crucial role in pollen wall pattern formation, tapetum development, and auxin signal transduction in anthers. Here, we showed that ARF17 is also involved in anther dehiscence. The Arabidopsis (Arabidopsis thaliana) arf17 mutant exhibits defective endothecium lignification, which leads to defects in anther dehiscence. The expression of MYB108, which encodes a transcription factor important for anther dehiscence, was dramatically down-regulated in the flower buds of arf17 Chromatin immunoprecipitation assays and electrophoretic mobility shift assays showed ARF17 directly binds to the MYB108 promoter. In an ARF17-GFP transgenic line, in which ARF17-GFP fully complements the arf17 phenotype, ARF17-GFP was observed in the endothecia at anther stage 11. The GUS signal driven by the MYB108 promoter was also detected in endothecia at late anther stages in transgenic plants expressing promoterMYB108::GUS Thus, the expression pattern of both ARF17 and MYB108 is consistent with the function of these genes in anther dehiscence. Furthermore, the expression of MYB108 driven by the ARF17 promoter successfully restored the defects in anther dehiscence of arf17 These results demonstrated that ARF17 regulates the expression of MYB108 for anther dehiscence. Together with its function in microcytes and tapeta, ARF17 likely coordinates the development of different sporophytic cell layers in anthers. The ARF17-MYB108 pathway involved in regulating anther dehiscence is also discussed.

Fine regulation of ARF17 for anther development and pollen formation.

BACKGROUND: In Arabidopsis, the tapetum and microsporocytes are critical for pollen formation. Previous studies have shown that ARF17 is expressed in microsporocytes and tetrads and directly regulates tetrad wall synthesis for pollen formation. ARF17 is the direct target of miR160, and promoterARF17::5mARF17 (5mARF17/WT) transgenic plants, which have five silent mutations within the miR160-complementary domain, are sterile. RESULTS: Here, we found that ARF17 is also expressed in the tapetum, which was defective in arf17 mutants. Compared with arf17 mutants, 5mARF17/WT plants had abnormal tapetal cells and tetrads but were less vacuolated in the tapetum. Immunocytochemical assays showed that the ARF17 protein over-accumulated in tapetum, microsporocytes and tetrads of 5mARF17/WT plants at early anther stages, but its expression pattern was not affected during anther development. 5mARF17 driven by its native promoter did not rescue the arf17 male-sterile phenotype. The expression of 5mARF17 driven by the tapetum-specific promoter A9 led to a defective tapetum and male sterility in transgenic plants. These results suggest that the overexpression of ARF17 in the tapetum and microsporocytes of 5mARF17/WT plants leads to male sterility. Microarray data revealed that an abundance of genes involved in transcription and translation are ectopically expressed in 5mARF17/WT plants. CONCLUSIONS: Our work shows that ARF17 plays an essential role in anther development and pollen formation, and ARF17 expression under miR160 regulation is critical for its function during anther development.

Polymeric phenylpropanoid derivatives crosslinked by hydroxyl fatty acids form the core structure of rape sporopollenin.

Sporopollenin, a critical innovation in the evolution of terrestrial plants, is the core building brick for the outer wall of land-plant spores and pollen. Despite its significance, the basic structure of sporopollenin remains elusive due to its extreme chemical inertness. In this study, we used ethanolamine to completely dissolve rape sporopollenin and successfully identified a total of 22 components, including fatty acids, p-coumaric acid, sterols and polymeric phenylpropanoid derivatives. After that, using NaOH treatment and partial dissolution, alongside Arabidopsis mutants analysis and spectroscopic methods, we determined that polymeric phenylpropanoid derivatives crosslinked by hydroxyl fatty acids serve as the core structure of sporopollenin. The free hydroxyl groups and carboxyl groups of the polymeric phenylpropanoid derivatives can be modified by other fatty acids (C16:0, C18:0 and C18:3) as well as alcohols/phenols (for example, naringenin, ß-sitosterol), resulting in a structure that protects pollen from terrestrial stresses. This discovery provides a basis for further exploration of sporopollenin's role in plant reproduction and evolution.

Documenting the Sporangium Development of the Polypodiales Fern Pteris multifida.

Reconstructing the development of sporangia in seed-free vascular plants provides crucial information about key processes enabling the production of spores that are important in the life cycle of these plants. By applying fluorescence imaging in intact tissues using dyes and confocal microscopy, this study aimed to reconstruct the key steps during the development of sporangia. Special emphasis was taken on the cell wall structures of tapetum and spore mother cells that have been challenged by microscopical documentation in the past. After staining the cell wall and cytoplasm using calcofluor white and basic fuchsin, the sporangium development of Pteris multifida was observed using confocal microscopy. The clear cell lineages from the sporangial initial cell to stalk, epidermis, inner tapetum, outer tapetum, and sporogenous cells were revealed by confocal imaging. The sporangium development improved in this work will be useful for a general understanding of fern spore formation.

A dye combination for the staining of pollen coat and pollen wall.

The pollen coat, which forms on the pollen surface, consists of a lipid-protein matrix. It protects pollen from desiccation and is involved in adhesion, pollen-stigma recognition, and pollen hydration during interactions with the stigma. The classical methods used for pollen coat observation are scanning and transmission electron microscopy. In this work, we screened a collection of fluorescence dyes and identified two fluorescent brighteners FB-52 and FB-184. When they were used together with the exine-specific dye, Basic fuchsin, the pollen coat and the exine structures could be clearly visualized in the pollen of Brassica napus. This co-staining method was applied successfully in staining pollen from Fraxinus chinensis, Calystegia hederacea, and Petunia hybrida. Using this method, small pollen coat-containing cavities were detected in the outer pollen wall layer of Oryza sativa and Zea mays. We further showed these dyes are compatible with fluorescent protein markers. In the Arabidopsis thaliana transgenic line of GFP-tagged pollen coat protein GRP19, GRP19-GFP was observed to form particles at the periphery of pollen coat. This simple staining method is expected to be widely used for the studies of the palynology as well as the pollen-stigma interaction.

Development of the Middle Layer in the Anther of Arabidopsis.

The middle layer is an essential cell layer of the anther wall located between the endothecium and tapetum in Arabidopsis. Based on sectioning, the middle layer was found to be degraded at stage 7, which led to the separation of the tapetum from the anther wall. Here, we established techniques for live imaging of the anther. We created a marker line with fluorescent proteins expressed in all anther layers to study anther development. Several staining methods were used in the intact anthers to study anther cell morphology. We clarified the initiation, development, and degradation of the middle layer in Arabidopsis. This layer is initiated from both the inner and outer secondary parietal cells at stage 4, stopped cell division at stage 6, and finally degraded at stage 11. The neighboring cell layers, the epidermis, and endothecium continued cell division until stage 10, which led to a thin middle layer. The degradation of the tapetum cell wall at stage 7 lead to its isolation from the anther wall. This work presents fundamental information on the development of the middle layer, which facilitates the further investigation of anther development and plant fertility. These live imaging methods could be useful in future studies.

Phenylpropanoid Derivatives Are Essential Components of Sporopollenin in Vascular Plants.

The outer wall of pollen and spores, namely the exine, is composed of sporopollenin, which is highly resistant to chemical reagents and enzymes. In this study, we demonstrated that phenylpropanoid pathway derivatives are essential components of sporopollenin in seed plants. Spectral analyses showed that the autofluorescence of Lilium and Arabidopsis sporopollenin is similar to that of lignin. Thioacidolysis and NMR analyses of pollen from Lilium and Cryptomeria further revealed that the sporopollenin of seed plants contains phenylpropanoid derivatives, including p-hydroxybenzoate (p-BA), p-coumarate (p-CA), ferulate (FA), and lignin guaiacyl (G) units. The phenylpropanoid pathway is expressed in the tapetum in Arabidopsis, consistent with the fact that the sporopollenin precursor originates from the tapetum. Further germination and comet assays showed that this pathway plays an important role in protection of pollen against UV radiation. In the pteridophyte plant species Ophioglossum vulgatum and Lycopodium clavata, phenylpropanoid derivatives including p-BA and p-CA were also detected, but G units were not. Taken together, our results indicate that phenylpropanoid derivatives are essential for sporopollenin synthesis in vascular plants. In addition, sporopollenin autofluorescence spectra of bryophytes, such as Physcomitrella and Haplocladium, exhibit distinct characteristics compared with those of vascular plants, indicating the diversity of sporopollenin among land plants.

Gene Regulatory Network for Tapetum Development in Arabidopsis thaliana.

In flowering plants, male gametophyte development occurs in the anther. Tapetum, the innermost of the four anther somatic layers, surrounds the developing reproductive cells to provide materials for pollen development. A genetic pathway of DYT1-TDF1-AMS-MS188 in regulating tapetum development has been proven. Here we used laser microdissection and pressure catapulting to capture and analyze the transcriptome data for the Arabidopsis tapetum at two stages. With a comprehensive analysis by the microarray data of dyt1, tdf1, ams, and ms188 mutants, we identified possible downstream genes for each transcription factor. These transcription factors regulate many biological processes in addition to activating the expression of the other transcription factor. Briefly, DYT1 may also regulate early tapetum development via E3 ubiquitin ligases and many other transcription factors. TDF1 is likely involved in redox and cell degradation. AMS probably regulates lipid transfer proteins, which are involved in pollen wall formation, and other E3 ubiquitin ligases, functioning in degradating proteins produced in previous processes. MS188 is responsible for most cell wall-related genes, functioning both in tapetum cell wall degradation and pollen wall formation. These results propose a more complex gene regulatory network for tapetum development and function.