The zinc-finger transcription factor BcMF20 and its orthologs in Cruciferae which are required for pollen development.

Brassica campestris Male Fertility 20 (BcMF20) is a typical zinc-finger transcription factor that was previously isolated from flower buds of Chinese cabbage (Brassica campestris ssp. chinensis). By applying expression pattern analysis, it can be known that BcMF20 was specifically and strongly expressed in tapetum and pollen, beginning from the uninucleate stage, and was maintained during the mature-pollen stage. As BcMF20 was highly conserved in Cruciferae, it can be indicated that this zinc-finger transcription factor is important during the growth of Cruciferae. In this study, 12 C2H2-type zinc-finger TFs which shared high homology with BcMF20 were found from NCBI via BLAST. A new molecular phylogenetic tree was constructed by the comparison between BcMF20 and these 12 C2H2-type zinc-finger TFs with NJ method. By analyzing this phylogenetic tree, the evolution of BcMF20 was discussed. Then, antisense RNA technology was applied in the transgenesis of Arabidopsis thaliana to get the deletion mutants of BcMF20, so that its function during the pollen development can be identified. The results showed: BcMF20 are in the same clade with three genes from Arabidopsis. The inhibition of BcMF20 expression led to smaller amounts of and lower rate in germination of pollen and lower rate in fruit setting in certain transgenetic plants. This also led to the complete collapse of pollen grains. By SEM and TEM, pollen morphology and anther development processes were observed. In the middle uninucleate microspore stage, a relatively thin or even no primexine was formed in microspores. This may result in the malformation of the pollen wall and finally cause the deformity of pollens. Above all, it can be indicated that BcMF20 may act as a part of regulation mechanisms of TAZ1 and MS1. Together they play a role in a genetic pathway in the tapetum to act on proliferation of tapetal cells and keep the normal development of pollens.

BcMF11, a novel non-coding RNA gene from Brassica campestris, is required for pollen development and male fertility.

KEY MESSAGE : BcMF11 as a non-coding RNA gene has an essential role in pollen development, and might be useful for regulating the pollen fertility of crops by antisense RNA technology. We previously identified a 828-bp full-length cDNA of BcMF11, a novel pollen-specific non-coding mRNA-like gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). However, little information is known about the function of BcMF11 in pollen development. To investigate its exact biological roles in pollen development, the BcMF11 cDNA was antisense inhibited in transgenic Chinese cabbage under the control of a tapetum-specific promoter BcA9 and a constitutive promoter CaMV 35S. Antisense RNA transgenic plants displayed decreasing expression of BcMF11 and showed distinct morphological defects. Pollen germination test in vitro and in vivo of the transgenic plants suggested that inhibition of BcMF11 decreased pollen germination efficiency and delayed the pollen tubes' extension in the style. Under scanning electron microscopy, many shrunken and collapsed pollen grains were detected in the antisense BcMF11 transgenic Chinese cabbage. Further cytological observation revealed abnormal pollen development process in transgenic plants, including delayed degradation of tapetum, asynchronous separation of microspore, and aborted development of pollen grain. These results suggest that BcMF11, as a non-coding RNA, plays an essential role in pollen development and male fertility.

Spermidine affects the transcriptome responses to high temperature stress in ripening tomato fruit.

OBJECTIVE: High temperature adversely affects quality and yield of tomato fruit. Polyamine can alleviate heat injury in plants. This study is aimed to investigate the effects of polyamine and high temperature on transcriptional profiles in ripening tomato fruit. METHODS: An Affymetrix tomato microarray was used to evaluate changes in gene expression in response to exogenous spermidine (Spd, 1 mmol/L) and high temperature (33/27 °C) treatments in tomato fruits at mature green stage. RESULTS: Of the 10101 tomato probe sets represented on the array, 127 loci were differentially expressed in high temperature-treated fruits, compared with those under normal conditions, functionally characterized by their involvement in signal transduction, defense responses, oxidation reduction, and hormone responses. However, only 34 genes were up-regulated in Spd-treated fruits as compared with non-treated fruits, which were involved in primary metabolism, signal transduction, hormone responses, transcription factors, and stress responses. Meanwhile, 55 genes involved in energy metabolism, cell wall metabolism, and photosynthesis were down-regulated in Spd-treated fruits. CONCLUSIONS: Our results demonstrated that Spd might play an important role in regulation of tomato fruit response to high temperature during ripening stage.

Genotypic differences in pod wall and seed growth relate to invertase activities and assimilate transport pathways in asparagus bean.

BACKGROUND AND AIMS: Coordination of sugar transport and metabolism between developing seeds and their enclosing fruit tissues is little understood. In this study the physiological mechanism is examined using two genotypes of asparagus bean (Vigna unguiculata ssp. sesquipedialis) differing in pod wall and seed growth rates. Pod growth dominates over seed growth in genotype 'Zhijiang 121' but not in 'Zhijiang 282' in which a 'bulging pod' phenotype is apparent from 8 d post-anthesis (dpa) onward. METHODS: Seed and pod wall growth rates and degree of pod-bulging were measured in the two genotypes together with assays of activities of sucrose-degrading enzymes and sugar content in pod wall and seed and evaluation of cellular pathways of phloem unloading in seed coat using a symplasmic fluorescent dye, 5(6)-carboxyfluorescein (CF). KEY RESULTS: Activities of cell wall, cytoplasmic and vacuolar invertases (CWIN, CIN and VIN) were significantly smaller in pod walls of '282' than in '121' at 10 dpa onwards. Low INV activities were associated with weak pod wall growth of '282'. In seed coats, CF was confined within the vasculature in '282' but moved beyond the vasculature in '121', indicating apoplasmic and symplasmic phloem unloading, respectively. Higher CWIN activity in '282' seed coats at 6-8 dpa correlated with high hexose concentration in embryos and enhanced early seed growth. However, CWIN activity in '282' decreased significantly compared with '121' from 10 dpa onwards, coinciding with earlier commencement of nuclei endoreduplication in their embryos. CONCLUSIONS: The study shows genotypic differences between 'bulging pod' and 'non-bulging' phenotypes of asparagus bean in sucrose metabolism in relation to the pathway of phloem unloading in developing seed coats, and to pod and seed growth. Low INV activity in pod wall corresponds to its shortened and weak growth period; by contrast, the apoplasmic path in the seed coat is associated with high CWIN activity and strong early seed growth.

[The motive force of evolution based on the principle of organismal adjustment evolution.].

From the analysis of the existing problems of the prevalent theories of evolution, this paper discussed the motive force of evolution based on the knowledge of the principle of organismal adjustment evolution to get a new understanding of the evolution mechanism. In the guide of Schrodinger's theory - "life feeds on negative entropy", the author proposed that "negative entropy flow" actually includes material flow, energy flow and information flow, and the "negative entropy flow" is the motive force for living and development. By modifying my own theory of principle of organismal adjustment evolution (not adaptation evolution), a new theory of "regulation system of organismal adjustment evolution involved in DNA, RNA and protein interacting with environment" is proposed. According to the view that phylogenetic development is the "integral" of individual development, the difference of negative entropy flow between organisms and environment is considered to be a motive force for evolution, which is a new understanding of the mechanism of evolution. Based on such understanding, evolution is regarded as "a changing process that one subsystem passes all or part of its genetic information to the next generation in a larger system, and during the adaptation process produces some new elements, stops some old ones, and thereby lasts in the larger system". Some other controversial questions related to evolution are also discussed.

Characterization of a putative pollen-specific arabinogalactan protein gene, BcMF8, from Brassica campestris ssp. chinensis.

The BcMF8 (Brassica campestris male fertility 8) gene, possessing the features of 'classical' arabinogalactan protein (AGP) was isolated from Brassica campestris L. ssp. chinensis, Makino syn. B. rapa L. ssp. chinensis. This gene was highly abundant in the fertile flower buds but silenced in the sterile ones of genic male sterile A/B line ('ZUBajh97-01A/B') in B. campestris. Expression patterns analysis suggested BcMF8 was a pollen-specific gene, whose transcript started to be expressed at the uninucleate stage and maintained throughout to the pollen at pollination stage. BcMF8 is highly homologous to the known pollen-specific AGP genes Sta 39-4 and Sta 39-3 from B. napus. Isolation and multiple alignment of the homologs of BcMF8 gene in the family Cruciferae indicated that BcMF8 was highly conserved in this family, which reflect the conservation in biological function and importance of this putative AGP gene in plant development. Similarity analysis also demonstrated Sta 39-4 and Sta 39-3 may originate from different genomes.

[Molecular cloning and characterization of BcMYBogu, a novel member of the MYB family involved in OguCMS in Brassica campestris ssp. chinensis].

In the attempt to elucidate the molecular mechanism of CMS. Ogura cytoplasmic male sterile (OguCMS) lines were obtained in Chinese cabbage after interspecific hybridization between Brassica. napus L. OguCMS and B. campestris ssp. chinensis followed by recurrent backcross with B. campestris ssp. chinensis as the pollen donor. The CMS lines were significantly characterized by the whitish anther and indehiscence of anther. The tapetal hypertrophy with excess vacuola-tion was the first observed defective soon after the tetrad stage, subsequently the microspores defected in pollen wall forma-tion, and later the cytoplasm detached from the exine wall and underwent degeneration. With aid of cDNA-AFLP and RACE approaches, we cloned the BcMYBogu(GenBank accession No: EF127861) in Chinese cabbage, which is premature expressed in early and middle stage floral buds of OguCMS lines, and predicted to encode a novel protein with a DNA binding domain: SH[AL]QKY[RF] motif at the N-terminus. Phylogenetic comparison revealed that the BcMYBogu was clustered with AtMYB32, AtMYB26 and AtMYB4, which were indicated to be involved in male sterility in Arabidopsis thaliana. The BcMYBogu transcript was detected in rosette leaves, floral buds and stems by RT-PCR analysis. Compared with the maintainer, the expression level of BcMYBogu was increased in these organs, especially in floral buds of OguCMS lines. Our investigation suggests that BcMYBogu is a new member of the MYB family involved in male sterility in Chinese cabbage.

[Cloning and evolutionary analysis of homologous sequences of SAMDC gene in Cruciferae].

S-adenosylmethionine decarboxylase activity (SAMDC) is a key enzyme involved in biosynthesis of the polyamines, viz. spermidine and spermine. In the present study, SAMDC gene analogues from 14 species of 6 genera in Cruciferae were obtained by PCR strategy using specific primers designed from conserved regions of SAMDC gene reported in the GenBank. The phylogenetic relationships of these species belonging to the family Cruciferae were investigated through comparison of the genes. Homologous sequences of SAMDC comparison indicated that the similarities among the genes at nucleotide and amino acid levels were over 87% and 90%, respectively. The differences in genes at nucleotide and amino acid levels between species ranged from 0.2 % to 10.1 % and 0.3% to 6.6%, respectively, while those between genera except Raphanus were 4.9 % to 13.6 % and 3.1 % to 10.3%, respectively. The differences of sequences of nucleotides and amino acids among genera were higher than those among species, and the differences of nucleotides sequences were higher than those of amino acids. The phylogenetic tree was thus constructed based on the alignment nucleotides sequences from Nei's genetic distances. The neighbor-joining (NJ) and minimum-evolution (ME) trees showed that Brassica was closely related to Raphanus, followed by Barbarea, Roripl Scop and Arabidopsis Heynh, but was remotely related to Capsella Medic. This research elucidated the relationship among crucifer species at nucleotide level, which could proved some help for the utilization of plant germplasms.

BcMF11, a putative pollen-specific non-coding RNA from Brassica campestris ssp. chinensis.

A full-length cDNA, BcMF11, has been cloned from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) using rapid amplification of the cDNA ends (RACE) based on a pollen-specific cDNA fragment (DN237921). The BcMF11 cDNA has a total length of 828bp with poly (A) tail. Analysis of the sequence demonstrated that BcMF11 is a novel non-coding RNA which has no prominent open reading frame (ORF) or coding capacity. No significant similarities were observed between BcMF11 and previously published sequences in GenBank. Transcription analysis indicated that BcMF11 is a novel pollen-specific ncRNA and may be involved in the pollen development of Chinese cabbage.

Comparative transcript profiling by cDNA-AFLP reveals similar patterns of Avr4/Cf-4- and Avr9/Cf-9-dependent defence gene expression.

SUMMARY: Tomato Cf genes confer resistance to the fungal pathogen Cladosporium fulvum. Although the Cf-4 and Cf-9 proteins are very similar, the Cf-4- and Cf-9-dependent hypersensitive responses (HRs) are distinct in cell death pattern, intensity and sensitivity to environmental conditions. To investigate the mechanism leading to these differences, comparative transcript profiling of Avr4/Cf-4- and Avr9/Cf-9-dependent defence gene expression was performed. To do this, cDNA-AFLP analysis was conducted on Avr/Cf tomato seedlings undergoing early HR. Both Avr4/Cf-4 and Avr9/Cf-9 signalling elicited the same spectrum of genes, referred to here as Avr/Cf-elicited (ACE) genes. Of approximately 25 000 transcript-derived fragments (TDFs), 367 (1.5%) showed significant differential expression between HR(+) and HR(-) seedlings (either Avr4/Cf-4- or Avr9/Cf-9-dependent). However, 42.8% of the ACE TDFs (157/367 in total) showed quantitatively different expression in the two types of HR(+) seedlings. The majority of these (135/157, 86.0%) displayed significantly greater differential expression (either induced or repressed) in Avr4/Cf-4 HR(+) seedlings than in Avr9/Cf-9 HR(+) seedlings. Our results are consistent with the previous observation that Avr4/Cf-4-dependent HR is more severe than Avr9/Cf-9-dependent HR, and indicate that the distinction between Avr4/Cf-4- and Avr9/Cf-9-dependent HR is most probably a result of events upstream of the defence responses. Sequencing of 189 ACE fragments identified genes associated with: defence and resistance (33.3%), signal transduction (7.4%), HR and cell death (5.3%), transcriptional regulation and post-transcriptional modification (4.3%). Expression data revealed that defence response, respiration and biological oxidation are strongly induced while photosynthesis is severely repressed in the HR(+) seedlings.

[BcMF4 gene, encoding a leucine-rich repeat protein, plays a role in male fertility in Chinese cabbage-pak-choi].

The BcMF4 (Brassica campestris Male Fertility 4) gene was previously isolated from the fertile B line of Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis var. communis, syn. B. rapa ssp. chinensis var. communis). In the present paper, based on the cDNA sequence of BcMF4, primers were designed and used to amplify two fragments from the cDNA of flower buds of Chinese cabbage-pak-choi. Two produced fragments were introduced separately into binary vector pBI121 in antisense and sense orientations. The generated RNA interference (RNAi) vector was then mobilized into Agrobacterium tumefaciens strain LBA4404. The A. tumefaciens harboring the BcMF4 fragments was transformed to flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis) via tissue culture. Approximately 45.8% of the pollen grains from 72.2% of RNAi plants exhibited abnormal in their shapes, and only 23.7% of the pollen grains from these plants germinated normally. Northern blotting demonstrated that the phenotypic change of pollen grains resulted from the inhibition of expression of the BcMF4 due to the insertion of the transgene. This indicates that functional interrupting of BcMF4 by RNAi resulted in partial pollen abortion in flowering Chinese cabbage, suggesting that the product of BcMF4 gene plays an important role during pollen development of Chinese cabbage such as Chinese cabbage-pak-choi and flowering Chinese cabbage.

[Construction of an activation tagging library of Arabidopsis and cloning for mutant genes].

Activation tagging is an important strategy in plant genomics by generating gain-of-function mutants. In this work, a library of Arabidopsis mutants was constructed by in planta transformation mediated by Agrobacterium tumefaciens containing an activation tagging vector pSKI015 with herbicide Basta as a selection marker (Fig. 1). Among 20000 independent transformants, 38 lines, i.e. about 0.2% of T(1) progeny, show visible morphological phenotypic variations (Fig. 2). Results of Southern blot analysis revealed that most of the transformants have more than three copies of T-DNA insertion (Fig. 3). Plasmid rescue and TAIL-PCR were used to recover the flanking genomic sequences of mutated target genes as the first step towards mutant gene cloning (Fig. 4, 5).

[Cloning and Evolutionary Analysis of Homologous Sequences of CYP86MF Gene in Cruciferae.].

In order to direct the construction of plant germplasms by elucidating the relatives among plants at the level of gene, CYP86MF gene analogues from 11 species of 6 genera in Cuciferae were respectively obtained by PCR strategy using gene specific primers designed from conserved regions of CYP86MF gene reported. Sequence comparisonindicated that the similarities among the genes at nucleotide level were over 80%, and the similarities at amino acid level remained above 70%. The differences between the genes at nucleotide and amino acid level between species were 1.0% ~ 5.7% and 2.6% ~ 7.3% respectively, while those between genera 5.6% ~ 22.5% and 7.3% ~ 31.2%, respectively. Phylogenetic analysis showed that Brassica was closely related to Raphanus, followed by Rorippa Scop, Arabidopsis Heynh, Capsella Medic orderly, most distantly related to Orychophrogmus. It was concluded that CYP86MF gene was not applicable to specie and subspecie taxon but genus taxon because the differences of sequences in nucleotides and amino acids were lower between species than genera.

[Differences of gene expression in bud stage of backcross hybrid between Ogura-type male-sterile Brassica napus L and B campestris L versus parents].

Crosses between female parent of Ogura male sterility Brassica napus L. and male parents of B. campestris ssp. chinensis Makino were made and F(1), BC(1) and BC(2) generations produced. Gene expression of two Chinese cabbage backcross hybrid BC(1), BC(2) and their parents at bud stage was analyzed by means of cDNA-AFLP technique. The results indicated that the patterns of gene expression differ significantly between BC(1) and BC(2) generations and their parents. There were many patterns of gene expression, including gene overexpression and gene silencing. Five patterns (seven kinds) of gene expression were observed, which include: (1) bands occurring in only one parent (two kinds); (2) bands observed in hybrids and one parent (two kinds); (3) bands occurring in only parents (one kind); (4) bands visualized in only hybrids (one kind); (5) bands observed in parents and hybrids (one kind). In accompany with the addition of backcross, the increase trend in backcross hybrids and their parents were described in the aspects of differential gene expression, bands expressed only in one parent and bands expressed only in both parents. The declined trend in backcross hybrids and their parents were observed in the aspects of bands expressed in both hybrids and one parent (two kinds), bands visualized in only hybrids and bands observed in parents and hybrid. Fifteen patterns of gene expression were observed in F(1)bBC(1)bBC(2) and backcross parents. The percent of bands expressed in F(1)bBC(1)bBC(2) and backcross was highest.

[sdt1, a novel Arabidopsis mutant tolerant to drought and high-salinity stresses].

Among the 20000 independent Arabidopsis activation tagging lines, we screened a novel mutant with high-salinity and drought tolerance, namely sdt1 (high-salinity and drought tolerant 1). sdt1 kept normal growth under drought stress by consecutively withholding water for 26d, while all the wild type wilted to death. Furthermore, the same result was repeated under high-salinity stress when sdtl and wild type were treated with 150 mmol/L NaCl. Seeds germination test on the medium indicated that the response of sdt1 to endogenous or exogenous ABA was reduced as compared with wild type. Under drought stress conditions the rates of water loss of sdt1 rosette leaves were significantly lower than that of wild type. Southern hybridization of sdt1 and the result of high-salinity tolerance genetic analysis showed that two copies of T-DNA were inserted into the two linking sites by shoulder to shoulder.

[Establishment of an efficient regeneration system for Chinese cabbage cultured in vitro and histological observation of shoot development].

An efficient plant regeneration system from explants of petiole with cotyledon was developed for Chinese cabbage (Brassica campestris ssp. chinensis Makino) in our study. High frequency of shoot regeneration was obtained in the medium containing BAP 2 mg/L, NAA 0.45 mg/L and 5-7.5 mg/L AgNO3 solution to the half of NH4+ concentration's MS basic medium, with 84.3 percent being the highest frequency of shoot regeneration in seven Chinese cabbage cultivars and one parentage, and the new formed shoots were observed six days after inoculation. Factors influencing in vitro explant regeneration were further studied. The results showed that there was positive correlation between frequency of shoot regeneration and number of shoots per explant. The regeneration plants produced flowers and fruits after about 50 days with low temperature treatment from 8 degrees C to 16 degrees C. Moreover, histological observation indicated that the mode of plant regeneration in B. campestris ssp. chinensis was similar to that of other Brassica crops, and adventitions buds were formed from globular meristematic cluster which derived from the cells of vascular parenchyma at the cut surface of petiole.