A cellular mechanism underlying the restoration of thermo/photoperiod-sensitive genic male sterility.

During anther development, the transformation of the microspore into mature pollen occurs under the protection of first the tetrad wall and later the pollen wall. Mutations in genes involved in this wall transition often lead to microspore rupture and male sterility; some such mutants, such as the reversible male sterile (rvms) mutant, are thermo/photoperiod-sensitive genic male sterile (P/TGMS) lines. Previous studies have shown that slow development is a general mechanism of P/TGMS fertility restoration. In this study, we identified restorer of rvms-2 (res2), which is an allele of QUARTET 3 (QRT3) encoding a polygalacturonase that shows delayed degradation of the tetrad pectin wall. We found that MS188, a tapetum-specific transcription factor essential for pollen wall formation, can activate QRT3 expression for pectin wall degradation, indicating a non-cell-autonomous pathway involved in the regulation of the cell wall transition. Further assays showed that a delay in degradation of the tetrad pectin wall is responsible for the fertility restoration of rvms and other P/TGMS lines, whereas early expression of QRT3 eliminates low temperature restoration of rvms-2 fertility. Taken together, these results suggest a likely cellular mechanism of fertility restoration in P/TGMS lines, that is, slow development during the cell wall transition of P/TGMS microspores may reduce the requirement for their wall protection and thus support their development into functional pollens, leading to restored fertility.

MS1, a direct target of MS188, regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis.

Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen-stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP-oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1.

The temporal regulation of TEK contributes to pollen wall exine patterning.

Pollen wall consists of several complex layers which form elaborate species-specific patterns. In Arabidopsis, the transcription factor ABORTED MICROSPORE (AMS) is a master regulator of exine formation, and another transcription factor, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), specifies formation of the nexine layer. However, knowledge regarding the temporal regulatory roles of TEK in pollen wall development is limited. Here, TEK-GFP driven by the AMS promoter was prematurely expressed in the tapetal nuclei, leading to complete male sterility in the pAMS:TEK-GFP (pat) transgenic lines with the wild-type background. Cytological observations in the pat anthers showed impaired callose synthesis and aberrant exine patterning. CALLOSE SYNTHASE5 (CalS5) is required for callose synthesis, and expression of CalS5 in pat plants was significantly reduced. We demonstrated that TEK negatively regulates CalS5 expression after the tetrad stage in wild-type anthers and further discovered that premature TEK-GFP in pat directly represses CalS5 expression through histone modification. Our findings show that TEK flexibly mediates its different functions via different temporal regulation, revealing that the temporal regulation of TEK is essential for exine patterning. Moreover, the result that the repression of CalS5 by TEK after the tetrad stage coincides with the timing of callose wall dissolution suggests that tapetum utilizes temporal regulation of genes to stop callose wall synthesis, which, together with the activation of callase activity, achieves microspore release and pollen wall patterning.

Slowing development restores the fertility of thermo-sensitive male-sterile plant lines.

Temperature-sensitive genic male sterility (TGMS) lines are widely used in the breeding of hybrid crops1,2, but by what means temperature as a general environmental factor reverses the fertility of different TGMS lines remains unknown. Here, we identified an Arabidopsis TGMS line named reversible male sterile (rvms) that is fertile at low temperature (17 °C) and encodes a GDSL lipase. Cytological observations and statistical analysis showed that low temperature slows pollen development. Further screening of restorers of rvms, as well as crossing with a slow-growth line at normal temperature (24 °C), demonstrate that slowing of development overcomes the defects of rvms microspores and allows them to develop into functional pollen. Several other Arabidopsis TGMS lines were identified, and their fertility was also restored by slowing of development. Given that male reproductive development is conserved3, we propose that slowing of development is a general mechanism applicable to the sterility-fertility conversion of TGMS lines from different plant species.

A stable nanoscaled Zr-MOF for the detection of toxic mycotoxin through a pH-modulated ratiometric luminescent switch.

A stable nanoscaled single-excitation ratiometric luminescent pH sensor (MPDB-PCN) over a broad pH range from 2.5 to 8.6 is fabricated through post-synthetic modification of PCN-224 with naphthalimide-derived molecules. Due to the rapid, sensitive and linear response to pH, MPDB-PCN is capable of detecting 3-nitropropionic acid (3-NPA), an acid neurotoxin in food safety, with a low detection limit of 15 µM in sugarcane juice.

Acyl-CoA synthetases from Physcomitrella, rice and Arabidopsis: different substrate preferences but common regulation by MS188 in sporopollenin synthesis.

MAIN CONCLUSION: ACOS5, OsACOS12 and PpACOS6 are all capable of fatty acyl-CoA synthetase activity but exhibit different substrate preferences. The transcriptional regulation of ACOS for sporopollenin synthesis appears to have been conserved in Physcomitrella, rice and Arabidopsis during evolution. Sporopollenin is the major constituent of spore and pollen exines. In Arabidopsis, acyl-CoA synthetase 5 (ACOS5) is an essential enzyme for sporopollenin synthesis, and its orthologues are PpACOS6 from the moss Physcomitrella and OsACOS12 from monocot rice. However, knowledge regarding the evolutionary conservation and divergence of the ACOS gene in sporopollenin synthesis remains limited. In this study, we analysed the function and regulation of PpACOS6 and OsACOS12. A complementation test showed that OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis, while PpACOS6 did not rescue the acos5 phenotype. ACOS5, PpACOS6 and OsACOS12 all complemented the acyl-CoA synthetase-deficient yeast strain (YB525) phenotype, although they exhibited different substrate preferences. To understand the conservation of sporopollenin synthesis regulation, we constructed two constructs with ACOS5 driven by the OsACOS12 or PpACOS6 promoter. Both constructs could restore the fertility of acos5 plants. The MYB transcription factor MS188 from Arabidopsis directly regulates ACOS5. We found that MS188 could also bind the promoters of OsACOS12 and PpACOS6 and activate the genes driven by the promoters, suggesting that the transcriptional regulation of these genes was similar to that of ACOS5. These results show that the ACOS gene promoter region from Physcomitrella, rice and Arabidopsis has been functionally conserved during evolution, while the chain lengths of fatty acid-derived monomers of sporopollenin vary in different plant species.

The Regulation of Sporopollenin Biosynthesis Genes for Rapid Pollen Wall Formation.

Sporopollenin is the major component of the outer pollen wall (sexine). It is synthesized using a pathway of approximately eight genes in Arabidopsis (Arabidopsis thaliana). MALE STERILITY188 (MS188) and its direct upstream regulator ABORTED MICROSPORES (AMS) are two transcription factors essential for tapetum development. Here, we show that all the sporopollenin biosynthesis proteins are specifically expressed in the tapetum and are secreted into anther locules. MS188, a MYB transcription factor expressed in the tapetum, directly regulates the expression of POLYKETIDE SYNTHASE A (PKSA), PKSB, MALE STERILE2 (MS2), and a CYTOCHROME P450 gene (CYP703A2). By contrast, the expression of CYP704B1, ACYL-COA SYNTHETASE5 (ACOS5), TETRAKETIDE a-PYRONE REDUCTASE1 (TKPR1) and TKPR2 are significantly reduced in ams mutants but not affected in ms188 mutants. However, MS188 but not AMS can activate the expression of CYP704B1, ACOS5, and TKPR1 In ms188, dominant suppression of MS188 homologs reduced the expression of these genes, suggesting that MS188 and other MYB family members play redundant roles in activating their expression. The expression of some sporopollenin synthesis genes (PKSA, PKSB, TKPR2, CYP704B1, and ACOS5) was rescued when MS188 was expressed in ams Therefore, MS188 is a key regulator for activation of sporopollenin synthesis, and AMS and MS188 may form a feed-forward loop that activates the expression of the sporopollenin biosynthesis pathway for rapid pollen wall formation.

The transcription factors MS188 and AMS form a complex to activate the expression of CYP703A2 for sporopollenin biosynthesis in Arabidopsis thaliana.

The sexine layer of pollen grain is mainly composed of sporopollenins. The sporophytic secretory tapetum is required for the biosynthesis of sporopollenin. Although several enzymes involved in sporopollenin biosynthesis have been reported, the regulatory mechanism of these enzymes in tapetal layer remains elusive. ABORTED MICROSPORES (AMS) and MALE STERILE 188/MYB103/MYB80 (MS188/MYB103/MYB80) are two tapetal cell-specific transcription factors required for pollen wall formation. AMS functions upstream of MS188. Here we report that AMS and MS188 target the CYP703A2 gene, which is involved in sporopollenin biosynthesis. We found that AMS and MS188 were localized in tapetum while CYP703A2 was localized in both tapetum and locule. Chromatin immunoprecipitation (ChIP) showed that MS188 directly bound to the promoter of CYP703A2 and luciferase-inducible assay showed that MS188 activated the expression of CYP703A2. Yeast two-hybrid and electrophoretic mobility shift assays (EMSAs) further demonstrated that MS188 complexed with AMS. The expression of CYP703A2 could be partially restored by the elevated levels of MS188 in the ams mutant. Therefore, our data reveal that MS188 coordinates with AMS to activate CYP703A2 in sporopollenin biosynthesis of plant tapetum.

Arabidopsis AT-hook protein TEK positively regulates the expression of arabinogalactan proteins for Nexine formation.

Nexine is a conserved layer of the pollen wall. We previously reported that the nexine layer is absent in the knockout mutant of Arabidopsis TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK) gene. In this study, we investigated the molecular regulatory functions of TEK in pollen development and identified the genes encoding Arabinogalactan proteins (AGPs) as direct targets of TEK, which are essential for nexine formation. Phenotypic similarity between tek and the TEK-SRDX transgenic lines suggest that TEK plays a role in transcriptional activation in anther development. Microarray analysis identified a total of 661 genes downregulated in tek, including four genes encoding AGPs, AGP6, AGP11, AGP23, and AGP40. Electrophoretic mobility shift assays showed that TEK could directly bind the nuclear matrix attachment region (MAR) and the promoter of AGP6. Chromatin immunoprecipitation followed by PCR analysis demonstrated that TEK is enriched in the promoters of the four AGP genes. Expression of AGP6 driven by the TEK promoter in tek partially rescued both nexine formation and plant fertility. These results indicate that TEK directly regulates AGP expression in the anther to control nexine layer formation. We also proposed that glycoproteins might be essential components of the nexine layer in the pollen wall.

DYT1 directly regulates the expression of TDF1 for tapetum development and pollen wall formation in Arabidopsis.

The tapetum plays a critical role during the development and maturation of microspores. DYSFUNCTIONAL TAPETUM 1 (DYT1) is essential for early tapetal development. Here, we determined that the promoter region (-550 to -463 bp) contains indispensable cis-elements for DYT1 expression. Although DYT1 transcripts can be detected in both meiocytes and tapetal cells, localization of DYT1-GFP demonstrated that DYT1 is strictly located in tapetal cells during microsporogenesis. Chromatin immunoprecipitation (ChIP) analysis revealed that DYT1 directly binds the promoter region of Defective in Tapetal Development and Function 1 (TDF1), a transcription factor essential for tapetum development. When TDF1 driven by the DYT1 promoter is expressed in a dyt1 mutant, the expression of the transcription factors AMS, MS188/MYB80, TEK and MS1 and the pollen wall-related genes are restored. Although the pollen wall is not formed and the microspores are ruptured, DIOC2 staining showed that fatty acids, the precursors of the pollen wall, were synthesized in the transgenic lines. These results indicate that DYT1 regulates the expression of AMS, MS188/MYB80, TEK and MS1 for pollen wall formation, primarily via TDF1.

The tapetal AHL family protein TEK determines nexine formation in the pollen wall.

The pollen wall, an essential structure for pollen function, consists of two layers, an inner intine and an outer exine. The latter is further divided into sexine and nexine. Many genes involved in sexine development have been reported, in which the MYB transcription factor Male Sterile 188 (MS188) specifies sexine in Arabidopsis. However, nexine formation remains poorly understood. Here we report the knockout of TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK) leads to nexine absence in Arabidopsis. TEK encodes an AT-hook nuclear localized family protein highly expressed in tapetum during the tetrad stage. Absence of nexine in tek disrupts the deposition of intine without affecting sexine formation. We find that ABORTED MICROSPORES directly regulates the expression of TEK and MS188 in tapetum for the nexine and sexine formation, respectively. Our data show that a transcriptional cascade in the tapetum specifies the development of pollen wall.

[Study on changes in cytokines of infertile women with mycoplasma infection and intervention with traditional Chinese medicines].

OBJECTIVE: To investigate the changes in cytokines (IL-1beta, IL-2, TNF-alpha) of peripheral blood and cervical mucous of infertile women with mycoplasma infection and the effect of intervention of traditional Chinese medicines (TCMs). METHOD: According to the results of culture of mycoplasma from genital tracts, 72 patients with positive mycoplasma were randomly divided into the TCM group (38 cases) and the western medicine group (34 cases). The western medicine group was treated with 0.5 g azithromycin for 3 days and consecutively treated for six courses of treatment, each course of treatment of 4 days. The TCM group were treated with Xiaozhi decoction twice every day for 6 weeks. The IL-1beta, IL-2 and TNF-alpha levels of the peripheral blood and cervical mucous of the two groups were measured by the Ria testing before and after the treatment, and the mycoplasma culture (-) of 32 infertile women as set for control. RESULT: Before the treatment, TNF-alpha and IL-1beta in levels of the two treatment groups were higher than those of the control group (P < 0.01). In the TCM group, TNF-alpha and IL-1beta levels showed significant differences compared with those before the treatment (P < 0.05) and those of the western group after the treatment (P < 0.01); and IL-2 level didn't have significant change before and after the treatment. The cytokines in peripheral blood of the two treatment groups showed notable difference compared with those of the control group (P < 0.01). In TCM group, IL-2 level had remarkable difference compared with that before the treatment (P < 0.01) and compared with the control group after the treatment (P < 0.01). CONCLUSION: Cytokines (IL-1beta, IL-2, TNF-alpha) in the peripheral blood and cervical mucous increase in infertile women with the mycoplasma infection, suggesting that TCMs can effectively inhibit the levels of IL-1beta, IL-2, TNF-alpha in the peripheral blood and IL-1beta, TNF-alpha in cervical mucous. It is proved that Xiaozhi decoction can be used to treat infertile women with mycoplasma infection.

A genetic pathway for tapetum development and function in Arabidopsis.

In anther development, tapetal cells take part in complex processes, including endomitosis and apoptosis (programmed cell death). The tapetum provides many of the proteins, lipids, polysaccharides and other molecules necessary for pollen development. Several transcription factors, including DYT1, TDF1, AMS, MS188 and MS1, have been reported to be essential for tapetum development and function in Arabidopsis thaliana. Here, we present a detailed cytological analysis of knockout mutants for these genes, along with an in situ RNA hybridization experiment and double mutant analysis showing that these transcription factors form a genetic pathway in tapetum development. DYT1, TDF1 and AMS function in early tapetum development, while MS188 and MS1 are important for late tapetum development. The genetic pathway revealed in this work facilitates further investigation of the function and molecular mechanisms of tapetum development in Arabidopsis.