ao-ao Ji

Mitochondrial cytochrome c oxidase and F1Fo-ATPase dysfunction in peppers (Capsicum annuum L.) with cytoplasmic male sterility and its association with orf507 and Ψatp6-2 genes.

Cytoplasmic male sterility (CMS) in pepper (Capsicum annuum L.) has been associated with novel genes in the mitochondria, such as orf507 and Ψatp6-2. Plant sterility has been proved to result from the rearrangement of the mitochondrial genome. Previous studies have demonstrated that orf507 is co-transcribed with the cox II gene, and Ψatp6-2 is truncated at the 3′ region of the atp6-2 that is found in the maintainer line. Until this time, little has been known about the relationship between the novel gene and the function of its corresponding enzyme in mitochondria from the CMS pepper line. Moreover, the aberrant function of the mitochondrial enzymes is seldom reported in pepper. In this study, we observed that anther abortion occurred after the tetrad stage in the CMS line (HW203A), which was accompanied by premature programmed cell death (PCD) in the tapetum. The spatiotemporal expression patterns of orf507 and Ψatp6-2 were analyzed together with the corresponding enzyme activities to investigate the interactions of the genes and mitochondrial enzymes. The two genes were both highly expressed in the anther. The orf507 was down-regulated in HW203A (CMS line), with nearly no expression in HW203B (the maintainer line). In contrast, the cytochrome c oxidase activity in HW203A showed the opposite trend, reaching its highest peak at the tetrad stage when compared with HW203B at the same stage. The Ψatp6-2 in the CMS line was also down-regulated, but it was up-regulated in the maintainer line. The corresponding F1Fo-ATPase activity in the CMS line was gradually decreased along with the development of the anther, which showed the same trend for Ψatp6-2 gene expression. On the contrary, with up-regulated gene expression of atp6-2 in the maintainer line, the F1Fo-ATPase activity sharply decreased after the initial development stage, but gradually increased following the tetrad stage, which was contrary to what happened in the CMS line. Taken together, all these results may provide evidence for the involvement of aberrant mitochondrial cytochrome c oxidase and F1Fo-ATPase in CMS pepper anther abortion. Moreover, the novel orf507 and Ψatp6-2 genes in the mitochondria may be involved in the dysfunction of the cytochrome c oxidase and F1Fo-ATPase, respectively, which are responsible for the abortion of anthers in the CMS line.

Cloning and expression analysis of heat-shock transcription factor gene CaHsfA2 from pepper (Capsicum annuum L.).

The heat-shock transcription factor (Hsf) gene CaHsfA2 (GenBank accession No. JX402923) was cloned from the Capsicum annuum thermotolerant line R9 by combining the techniques electron cloning and rapid amplification of cDNA ends. The gene, which is 1436 bp in length, had an open reading frame of 1089 bp that encoded 362 amino acids. There was an 831-bp intron between positions 321 and 322 of the cDNA. The deduced amino acid sequence of CaHsfA2 contained the conserved domains of Hsf, including DNA binding domain, adjacent domain with heptad hydrophobic repeats (A/B), activator motifs, nuclear localization signal, and nuclear export signal, and it had the highest E value of hypothesized annotation of HsfA2. CaHsfA2 had the nearest phylogenetic relationship with HsfA2 from Lycopersicon peruvianum and Mimulus guttatus, which was consistent with its botanical classification. After heat-shock treatment at 40°C for 2 h, the expression of CaHsfA2 was observed in different tissues of thermotolerant cultivar R9 and thermosensitive line B6; however, the expression levels of the CaHsfA2 gene were significantly different as follows: expression in B6 leaf > stem > flower > root, and expression in R9 flower > leaf > stem ≈ root.

Cloning and characterisation of a pepper aquaporin, CaAQP , which reduces chilling stress in transgenic tobacco plants

Ubiquitous cell membrane proteins called aquaporins are members of major intrinsic proteins (MIPs), which control the specific transport of water molecules across cell membranes. A pepper aquaporin gene (CaAQP), which exhibits the structural features of tonoplast intrinsic proteins of the MIP subfamily, was isolated from the leaves of chilling-treated seedlings of pepper (Capsicum annuum L.) cv. P70. Assays indicated high levels of expression in young seeds, green fruits and flower buds and low levels of expression in the stems, leaves and roots of pepper. The expression patterns were strongly and rapidly induced by HgCl2, low temperature, abscisic acid, fluridone and osmotic stresses. The responsiveness of pepper seedlings pretreated with abscisic acid at low temperatures demonstrated up-regulation of CaAQP by chilling, which is potentially involved in ABA signalling. Our results indicated that overexpression of CaAQP decreased chilling stress in transgenic plants, likely by increasing the stomatal aperture under stress, increasing the rate of membrane damage during the recovery stage, thereby affecting the intercellular CO2 concentration with lower stomatal conductance and transpiration rates. VIGS of CaAQP in pepper plants caused significant growth retardation. These results suggested that CaAQP played a crucial role in the plant response to abiotic stresses.

Tapetum-specific expression of a cytoplasmic orf507 gene causes semi-male sterility in transgenic peppers

Though cytoplasmic male sterility (CMS) in peppers is associated with the orf507 gene, definitive and direct evidence that it directly causes male sterility is still lacking. In this study, differences in histochemical localization of anther cytochrome c oxidase between the pepper CMS line and maintainer line were observed mainly in the tapetal cells and tapetal membrane. Inducible and specific expression of the orf507 gene in the pepper maintainer line found that transformants were morphologically similar to untransformed and transformed control plants, but had shrunken anthers that showed little dehiscence and fewer pollen grains with lower germination rate and higher naturally damaged rate. These characters were different from those of CMS line which does not produce any pollen grains. Meanwhile a pollination test using transformants as the male parent set few fruit and there were few seeds in the limited number of fruits. At the tetrad stage, ablation of the tapetal cell induced by premature programmed cell death (PCD) occurred in the transformants and the microspores were distorted and degraded at the mononuclear stage. Stable transmission of induced semi-male sterility was confirmed by a test cross. In addition, expression of orf507 in the maintainer lines seemed to inhibit expression of atp6-2 to a certain extent, and lead to the increase of the activity of cytochrome c oxidase and the ATP hydrolysis of the mitochondrial F1Fo-ATP synthase. These results introduce the premature PCD caused by orf507 gene in tapetal cells and semi-male sterility, but not complete male sterility.

Genetic determinants of the defense response of resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici (Oomycetes; Pythiaceae)

Based on culture isolation and morphological observation blight-infected pepper plants in Shaanxi Province, China, we identified the pathogen causing pepper phytophthora blight as Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602, and B27) were inoculated with this pathogen. The root activity of resistant CM334 variety was the highest while that of susceptible B27 variety was the lowest. Also, significant differences in the activity of POD, PAL, and β-1,3-glucanase were found; there was a positive correlation between disease resistance and activity of these three enzymes. We inhibited mycelial growth and sporangia formation of P. capsici using crude β-1,3-glucanase and PAL enzymes isolated from the resistant variety CM334 after it had been inoculated with P. capsici. These two enzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defensive genes CaPO1, CaBGLU, CaBPR1, and CaRGA in the three varieties was higher in the leaves than in the roots. All three genes were upregulated in infected leaves and roots of the pepper plants, always expressing at higher levels in the resistant cultivar than in the susceptible cultivar, suggesting that the differences in resistance among the pepper genotypes involve differences in the timing and magnitude of the defense response.

A CMS-Related Gene, Ψatp6-2, Causes Increased ATP Hydrolysis Activity of the Mitochondrial F1Fo-ATP Synthase and Induces Male Sterility in Pepper (Capsicum annuum L.)

Pollen formation is a complex process that is strictly controlled by genetic factors. Although many novel mitochondrial genes have been implicated in the dysfunction of mitochondrial enzymes and the cytoplasmic male sterility (CMS), there is little empirical evidence to show that CMS-related genes actually result in the dysfunction of enzyme and little is known about the regulatory mechanisms of the aberrant mitochondrial enzymes in male sterility in CMS lines. Here, we report the characterization of a novel mitochondrial gene, Ψatp6-2, which is hypothesized to play a role in male sterility in pepper. Using virus-induced gene silencing (VIGS), we observed that silencing the atp6-2 gene in the maintainer line resulted in an increase in ATP hydrolysis activity of the mitochondrial F1Fo-ATP synthase along with pollen abortion, while silencing the truncated Ψatp6-2 gene in the CMS line resulted in an inhibition of ATP hydrolysis activity and restoration of fertility. Altered ATP hydrolysis also affected the tolerance of the gene-silenced plants to abiotic stresses. Localization experiments showed that premature ATP hydrolysis occurred at the tetrad stage of pollen development in the CMS line, but no ATPase activity was observed in the microspores at the later stage. These results suggest that the Ψatp6-2 gene causes the alteration in ATP hydrolysis activity of the mitochondrial F1Fo-ATP synthase during pollen development, which eventually leads to male sterility in pepper.

Maintaining and restoring cytoplasmic male sterility systems in pepper (Capsicum annuum L.).

We studied the efficiency of maintaining and restoring cytoplasmic male sterility (CMS) systems in pepper (Capsicum annuum L.). An Rf-linked molecular marker was employed to analyze the interaction between 6 CMS lines (A), 5 maintainers (B), and 6 restorers (C). Sterility was maintained in the matings of lines 201A x 200B, 203A x 200B, 206A x 200B, 200A x 201B, 206A x 201B, 200A x 202B, 200A x 203B, 200A x 206B, and 201A x 206B. All 6 restorers restored the fertility of lines 200A, 202A, 203A, and 204A, except that 213C could not restore the fertility of lines 200A and 204A. However, the 6 restorers had diverse restoring abilities in individual CMS lines. The Rf-linked molecular marker was amplified by PCR in lines 207C, 208C, and 213C. This DNA marker was only found in the F1 hybrids M39, M14, M19, M25, M13, M20, and M22. We conclude that the restorers 208C and 207C can transmit the Rf gene or the Rf-linked marker to F1 hybrids.

Cloning and expression analysis of CaPIP1-1 gene in pepper (Capsicum annuum L.).

Plant aquaporins are responsible for water transmembrane transport, which play an important role on abiotic and biotic stresses. A novel plasma membrane intrinsic protein of CaPIP1-1 was isolated from the pepper P70 according to transcriptome databases of Phytophthora capsici inoculation and chilling stress library. CaPIP1-1, which is 1155 bp in length with an open reading frame of 861 bp, encoded 286 amino acids. Three introns, exhibited CT/AC splice junctions, were observed in CaPIP1-1. The numbers and location of introns in CaPIP1-1 were the same as observed in tomato and potato. CaPIP1-1 was abundantly expressed in pepper fruit. Increased transcription levels of CaPIP1-1 were found in the different stresses, including chilling stress, salt stress, mannitol stress, salicylic acid, ABA treatment and Phytophthora capsici infection. The expression of CaPIP1-1 was downregulated by 50 μM HgCl2 and 100 μM fluridone. The pepper plants silenced CaPIP1-1 in cv. Qiemen showed growth inhibition and decreased tolerance to salt and mannitol stresses using detached leaf method.

Novel genetic male sterility developed in (Capsicum annuum x C. chinense) x C. pubescens and induced by HNO2 showing Mendelian inheritance and aborted at telophase of microspore mother cell stage.

A novel genetic male sterile germplasm was developed by successively crossing of (C. annuum x C. chinense) x C. pubescens and by chemical mutagenesis in pepper. The sterile anthers showed morphological abnormalities, but pistils developed normally with fine pollination capability. We investigated fertility segregation through sib-crossing of the same strains and test crossing by male sterile plants with 6 advanced inbred lines. The results showed that male fertility in the pepper was dominant in the F1 generation and segregated at a rate of 3:1 in the F2 generation, suggesting that monogenic male sterility was recessive and conformed to Mendelian inheritance. Cyto-anatomy analysis revealed that microspore abortion of sterile anthers occurred during telophase in the microspore mother cell stage when tapetal cells showed excessive vacuolation, resulting in occupation of the loculi. The microspore mother cells self-destructed and autolyzed with the tapetum so that meiosis in pollen mother cells could not proceed past the tetrad stage.