[Nontargeted metabolomic analysis of Anoectochilus roxburghii at different cultivation stages].

Anoectochilus roxburghii is a traditional Chinese medicine and natural health products. In the modern cultivation system, A. roxburghii is micropropagated in tissue culture, and the plants are transferred to soil cultivation for months. However, it remains unclear about the necessity of soil cultivation for the accumulation of health beneficial compounds. In this paper, we performed nontargeted metabolomic analysis using GC-TOF-MS and UPLC-Q-TOF-MS, on A. roxburghii plants at tissue culture stage or after 3 months of soil cultivation. The results showed that the primary metabolites such as alcohols and organic acids are abundant in the tissue culture plants. In contrast, polysaccharide, nucleoside, esters and secondary metabolites such as flavonoids, terpenoids were significantly accumulated in cultivated seedlings. Flavonoids and polysaccharides are considered as the principle effective components in A. roxburghii. Soil cultivation period is therefore essential for the accumulation of these metabolites.

Arabidopsis RPG1 is important for primexine deposition and functions redundantly with RPG2 for plant fertility at the late reproductive stage.

Arabidopsis Ruptured Pollen Grain-1 (RPG1/Sweet8) is a member of the MtN3/saliva protein family that functions as a sugar transporter. The rpg1 mutant shows defective exine pattern formation. In this study, transmission electron microscopy (TEM) observations showed that much less primexine was deposited in rpg1 tetrads. Furthermore, microspore membrane undulation was abnormal, and sporopollenin accumulation was also defective. This suggests that a reduced primexine deposition in rpg1 leads to abnormal membrane undulation that affects exine pattern formation. Chemical staining revealed thinning of the callose wall of rpg1, as well as significantly reduced expression of Callose synthase-5 (CalS5) in rpg1. The fertility of the rpg1 mutant could be partly restored at late reproductive stages, potentially complemented in part by RPG2, another member of the MtN3/saliva family, which is expressed in the anther during microsporogenesis. The double mutant, rpg1rpg2, was almost sterile and was not restored during late reproduction. These results suggest that RPG1 and RPG2 are involved in primexine deposition and therefore pollen wall pattern formation.

AUXIN RESPONSE FACTOR17 is essential for pollen wall pattern formation in Arabidopsis.

In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression.

RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis.

During microsporogenesis, the microsporocyte (or microspore) plasma membrane plays multiple roles in pollen wall development, including callose secretion, primexine deposition, and exine pattern determination. However, plasma membrane proteins that participate in these processes are still not well known. Here, we report that a new gene, RUPTURED POLLEN GRAIN1 (RPG1), encodes a plasma membrane protein and is required for exine pattern formation of microspores in Arabidopsis (Arabidopsis thaliana). The rpg1 mutant exhibits severely reduced male fertility with an otherwise normal phenotype, which is largely due to the postmeiotic abortion of microspores. Scanning electron microscopy examination showed that exine pattern formation in the mutant is impaired, as sporopollenin is randomly deposited on the pollen surface. Transmission electron microscopy examination further revealed that the primexine formation of mutant microspores is aberrant at the tetrad stage, which leads to defective sporopollenin deposition on microspores and the locule wall. In addition, microspore rupture and cytoplasmic leakage were evident in the rpg1 mutant, which indicates impaired cell integrity of the mutant microspores. RPG1 encodes an MtN3/saliva family protein that is integral to the plasma membrane. In situ hybridization analysis revealed that RPG1 is strongly expressed in microsporocyte (or microspores) and tapetum during male meiosis. The possible role of RPG1 in microsporogenesis is discussed.

Defective in Tapetal development and function 1 is essential for anther development and tapetal function for microspore maturation in Arabidopsis.

In Arabidopsis, the tapetum plays important roles in anther development by providing enzymes for callose dissolution and materials for pollen-wall formation, and by supplying nutrients for pollen development. Here, we report the identification and characterization of a male-sterile mutant, defective in tapetal development and function 1 (tdf1), that exhibits irregular division and dysfunction of the tapetum. The TDF1 gene was characterized using a map-based cloning strategy, and was confirmed by genetic complementation. It encodes a putative R2R3 MYB transcription factor, and is highly expressed in the tapetum, meiocytes and microspores during anther development. Callose staining and gene expression analysis suggested that TDF1 may be a key component in controlling callose dissolution. Semi-quantitative and quantitative RT-PCR analysis showed that TDF1 acts downstream of DYT1 and upstream of AMS and AtMYB103 in the transcriptional regulatory networks that regulate tapetal development. In conclusion, our results show that TDF1 plays a vital role in tapetal differentiation and function.