Full-length EFOP3 and EFOP4 proteins are essential for pollen intine development in Arabidopsis thaliana.

Pollen development is critical to plant reproduction, but the underlying regulatory molecular mechanisms have not been fully elucidated. The Arabidopsis (Arabidopsis thaliana) EFR3 OF PLANT 3 (EFOP3) and EFR3 OF PLANT 4 (EFOP4) genes encode members of the Armadillo (ARM) repeat superfamily that play key roles in pollen development. Herein, we demonstrate that EFOP3 and EFOP4 are co-expressed in pollen at anther stages 10-12, but loss-of-function of both EFOP3 and EFOP4 leads to male gametophyte sterility, irregular intine, and shriveled pollen grains at anther stage 12. We further established that full-length EFOP3 and EFOP4 specifically localize to the plasma membrane, and the integrity of these proteins is essential for pollen development. We observed uneven intine, less organized cellulose and reduced pectin content in mutant pollen compared with the wild-type. These, together with the misexpression of several genes related to cell wall metabolism in efop3-/- efop4+/- mutants, suggest that EFOP3 and EFOP4 may indirectly regulate the expression of these genes to affect intine formation, thus controlling Arabidopsis pollen fertility in a functionally redundant manner. Moreover, transcriptome analysis showed that the absence of EFOP3 and EFOP4 function affects multiple pollen development pathways. These results enhance our understanding of EFOPs proteins and their role in pollen development.

POLLEN WALL ABORTION 1 is essential for pollen wall development in rice.

The integrity of pollen wall structures is essential for pollen development and maturity in rice (Oryza sativa L.). In this study, we isolated and characterized the rice male-sterile mutant pollen wall abortion 1 (pwa1), which exhibits a defective pollen wall (DPW) structure and has sterile pollen. Map-based cloning, genetic complementation, and gene knockout experiments revealed that PWA1 corresponds to the gene LOC_Os01g55094 encoding a coiled-coil domain-containing protein. PWA1 localized to the nucleus, and PWA1 was expressed in the tapetum and microspores. PWA1 interacted with the transcription factor TAPETUM DEGENERATION RETARDATION (TDR)-INTERACTING PROTEIN2 (TIP2, also named bHLH142) in vivo and in vitro. The tip2-1 mutant, which we obtained by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene editing, showed delayed tapetum degradation, sterile pollen, and DPWs. We determined that TIP2/bHLH142 regulates PWA1 expression by binding to its promoter. Analysis of the phenotype of the tip2-1 pwa1 double mutant indicated that TIP2/bHLH142 functions upstream of PWA1. Further studies suggested that PWA1 has transcriptional activation activity and participates in pollen intine development through the ß-glucosidase Os12BGlu38. Therefore, we identified a sterility factor, PWA1, and uncovered a regulatory network underlying the formation of the pollen wall and mature pollen in rice.

Oryza sativa PECTIN DEFECTIVE TAPETUM1 affects anther development through a pectin-mediated signaling pathway in rice.

Galacturonosyltransferase (GalAT) is required for the synthesis of pectin, an important component of plant cell walls that is also involved in signal transduction. Here, we describe the rice (Oryza sativa) male-sterile mutant O. sativa pectin-defective tapetum1 (ospdt1), in which GalAT is mutated. The ospdt1 mutant exhibited premature programmed cell death (PCD) of the tapetum and disordered pollen walls, resulting in aborted pollen grains. Pectin distribution in the anther sac was comparable between the mutant and the wild-type, suggesting that the structural pectin was not dramatically affected in ospdt1. Wall-associated kinases are necessary for the signal transduction of pectin, and the intracellular distribution of O. sativa indica WALL-ASSOCIATED KINASE1 (OsiWAK1), which binds pectic polysaccharides to its extracellular domain, was affected in ospdt1. OsiWAK1 RNA interference lines exhibited earlier tapetal PCD, similar to ospdt1. Furthermore, overexpression of OsiWAK1 in ospdt1 lines partially rescued the defects observed in ospdt1, suggesting that OsiWAK1 plays pivotal roles in the function of OsPDT1. These results suggest that the mutation of OsPDT1 does not dramatically affect structural pectin but affects components of the pectin-mediated signaling pathway, such as OsiWAK1, and causes male sterility.

OsMYB103 is essential for tapetum degradation in rice.

KEY MESSAGE: OsMYB103 positively regulates tapetum degradation, and functions downstream of TDR and upstream of EAT1 and PTC1. The precise regulation of programmed cell death (PCD) of the tapetum is crucial for the development of anthers and pollen in rice. In this study, we isolated and identified a male-sterile mutant of rice, osmyb103, which exhibited delayed tapetum degradation and defective mature pollen. Map-based cloning and genetic complementation revealed that OsMYB103 corresponded to the gene LOC_Os04g39470 and encoded a R2R3 MYB transcription factor. OsMYB103 was localized in the nucleus and was expressed preferentially in the tapetal cells and microspores of the anther. OsMYB103 regulated the expression of two transcription factors, ETERNAL TAPETUM 1 (EAT1) and PERSISTENT TAPETAL CELL 1 (PTC1), both of which regulated tapetum degradation positively. Moreover, the expression of OsMYB103 was directly regulated by the additional positive regulator of tapetum degradation TAPETUM DEGENERATION RETARDATION (TDR) and was able to interact with it. Genetic evidence confirmed that OsMYB103 acted upstream of EAT1. The results show that OsMYB103 is a positive regulator of tapetum degradation in rice. These findings provide a better understanding of the regulatory network that underlies degradation of the tapetum in rice.

OsFLA1 encodes a fasciclin-like arabinogalactan protein and affects pollen exine development in rice.

KEY MESSAGE: OsFLA1 positively regulates pollen exine development, and locates in the cellular membrane. Arabinogalactan proteins are a type of hydroxyproline-rich glycoprotein that are present in all plant tissues and cells and play important roles in plant growth and development. Little information is available on the participation of fasciclin-like arabinogalactan proteins in sexual reproduction in rice. In this study, a rice male-sterile mutant, osfla1, was isolated from an ethylmethanesulfonate-induced mutant library. The osfla1 mutant produced withered, shrunken, and abortive pollen. The gene OsFLA1 encoded a FLA protein and was expressed strongly in the anthers in rice. Subcellular localization showed that OsFLA1 was located in the cellular membrane. In the osfla1 mutant, abnormal Ubisch bodies and a discontinuous nexine layer of the microspore wall were observed, which resulted in pollen abortion and ultimately in male sterility. The results show the important role that OsFLA1 plays in male reproductive development in rice.

[Effect of nitrogen, phosphorus and potassium fertilizers on Flos Sophorae Immaturus yield and quality in hilly area of Chongqing].

With Sophora japonica at the flowering stage as the object, the effect of nitrogen, phosphorus and potassium fertilizers on the yield composition factors, yield and quality of Flos Sophorae Immaturus (FSI) was studied. The results indicated that in early spring, nitrogen, phosphorus and potassium fertilizer on the amplification rate of S. japonica, FSI yield composition, yield and quality were different significantly, middle to high nitrogen (1.5-2.0 kg/plant) significantly increased the level of panicled clusters, raceme and flower bud number and yield. Phosphorus (1.5-2.0 kg/plant) could significantly increase the total buds of flower number and yield, potassium showed no significant increase in yield and yield components. Comprehensively considering yield and quality of FSI, nitrogen 1.5-2.0 kg/plant, phosphorus 1.5-2.0 kg/plant and potassium 0.6-0.9 kg/plant are appropriate.

Optimization of Microwave-Assisted Extraction Conditions for Five Major Bioactive Compounds from Flos Sophorae Immaturus (Cultivars of Sophora japonica L.) Using Response Surface Methodology.

Microwave-assisted extraction was applied to extract rutin; quercetin; genistein; kaempferol; and isorhamnetin from Flos Sophorae Immaturus. Six independent variables; namely; solvent type; particle size; extraction frequency; liquid-to-solid ratio; microwave power; and extraction time were examined. Response surface methodology using a central composite design was employed to optimize experimental conditions (liquid-to-solid ratio; microwave power; and extraction time) based on the results of single factor tests to extract the five major components in Flos Sophorae Immaturus. Experimental data were fitted to a second-order polynomial equation using multiple regression analysis. Data were also analyzed using appropriate statistical methods. Optimal extraction conditions were as follows: extraction solvent; 100% methanol; particle size; 100 mesh; extraction frequency; 1; liquid-to-solid ratio; 50:1; microwave power; 287 W; and extraction time; 80 s. A rapid and sensitive ultra-high performance liquid chromatography method coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (EIS-Q-TOF MS/MS) was developed and validated for the simultaneous determination of rutin; quercetin; genistein; kaempferol; and isorhamnetin in Flos Sophorae Immaturus. Chromatographic separation was accomplished on a Kinetex C18 column (100 mm × 2.1 mm; 2.6 µm) at 40 °C within 5 min. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile (71:29; v/v). Isocratic elution was carried out at a flow rate of 0.35 mL/min. The constituents of Flos Sophorae Immaturus were simultaneously identified by EIS-Q-TOF MS/MS in multiple reaction monitoring mode. During quantitative analysis; all of the calibration curves showed good linear relationships (R² > 0.999) within the tested ranges; and mean recoveries ranged from 96.0216% to 101.0601%. The precision determined through intra- and inter-day studies showed an RSD% of <2.833%. These results demonstrate that the developed method is accurate and effective and could be readily utilized for the comprehensive quality control of Flos Sophorae Immaturus.

STAMENLESS 1, encoding a single C2H2 zinc finger protein, regulates floral organ identity in rice.

Floral organ identity is defined by organ homoetic genes whose coordinated expression is crucial with respect to the time and place of floral organ formation. Here, we report molecular cloning and characterization of the rice STAMENLESS 1 (SL1) gene that is involved in floral development. The sl1 mutant largely resembles the rice B-class gene mutant spw1; both exhibit homeotic conversions of lodicules and stamens to palea/lemma-like organs and carpels. Additionally, sl1 produces flowers with varied numbers of inner floral organs, and amorphous tissues without floral organ identity were frequently formed in whorls 3 and 4. We also show that SL1 specifies lodicule and stamen identities through positive transcriptional regulation of SPW1/OsMADS16 expression. SL1 encodes a member of the C2H2 family of zinc finger proteins, closely related to JAG of Arabidopsis. The functional divergence between SL1 and JAG implies that SL1 was co-opted for its distinctive roles in specification of floral organ identity in rice after the lineage split from Arabidopsis.

Genetic analysis and fine mapping of a dynamic rolled leaf gene, RL10(t), in rice (Oryza sativa L.).

A dynamically rolled leaf mutant (rl10) was identified from a spontaneous mutation in an Oryza sativa L. subsp. indica line, II-32B. The leaf chlorophyll content of rl10 is higher than that of the wild type. Genetic analysis using 3 F2 segregating populations derived from crosses between rl10 and the rice lines Mian5B, II-32B, and D62B, respectively, confirmed that the rolled leaf trait of rl10 is controlled by a single recessive gene. Of 719 SSR primer pairs that showed polymorphism between D62B and rl10, 151 were adopted to map the RL10(t) gene using an F2 segregating population of the cross rl10 x D62B, which contained 352 recessive plants. RL10(t) was primarily mapped on the long arm of chromosome 9, 5.09 cM from marker RM105 and 5.13 cM from marker RM3912. Using a novel set of 22 primer pairs between RM105 and RM3912, RL10(t) was further mapped between markers rlc3 (0.72 cM in distance) and rlc12 (0.1 cM in distance) using an F2/F3 population containing 1172 recessive individuals. Mapped position analysis and homology analysis of the 20 genes within the 194-kb region between these 2 markers both indicated that a gene encoding a Myb-like domain transcription factor with homology to Arabidopsis KANADI (annotated in PAC clone AP005904) is the most probable candidate for RL10(t). This study enables further investigation of whether KANADI-like Myb genes are involved in leaf polarity modeling in monocots, as they are in dicots.