Martha L. Crouch

Common amino acid sequence domains among the LEA proteins of higher plants

LEA proteins are late embryogenesis abundant in the seeds of many higher plants and are probably universal in occurrence in plant seeds. LEA mRNAs and proteins can be induced to appear at other stages in the plants life by desiccation stress and/or treatment with the plant hormone abscisic acid (ABA). A role in protecting plant structures during water loss is likely for these proteins, with ABA functioning in the stress transduction process. Presented here are conserved tracts of amino acid sequence among LEA proteins from several species that may represent domains functionally important in desiccation protection. Curiously, an 11 amino acid sequence motif is found tandemly repeated in a group of LEA proteins of vastly different sizes. Analysis of this motif suggests that it exists as an amphiphilic α helix which may serve as the basis for higher order structure.

Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.

Non-zygotic embryos of Brassica napus L. contain embryo-specific storage proteins

The storage-protein content of non-zygotic and zygotic embryos of B. napus was compared, using antibodies to guantitate 12S storage protein in extracts by rocket immunoelectrophoresis. Non-zygotic embryos were induced from microspores in anther culture and on the hypocotyls of zygotic embryos in culture. All embryo-like structures were found to contain 12S storage protein, whereas preculture anthers, anthers from which embryos had been removed, and regenerated shoots did not have detectable 12S storage protein. In zygotic embryos, 12S storage protein was first detected at the cotyledon stage, but microsporic embryos contained storage protein at the globular and heart stages. Storage protein levels in microsporic and hypocotyl embryos were low relative to those in zygotic embryos. The largest microsporic embryo had a storage protein concentration of 13 μg mg-1 fresh weight, almost 10 times lower than a mature zygotic embryo. Thus, although storage proteins are present in both zygotic and non-zygotic embryos, the timing and extent of accumulation differ.

Nucleotide sequence of a cDNA clone of Brassica napus 12S storage protein shows homology with legumin from Pisum sativum

SummaryThe most abundant protein in seeds of Brassica napus (L.) is cruciferin, a legumin-like 12S storage protein. By in vitro translation of embryo RNA, and pulse-chase labelling of cultured embryos with 14C-leucine, we have shown that the 30 kd α polypeptides and 20 kd β polypeptides of cruciferin are synthesized as a family of 50 kd precursors which are cleaved post-translationally. One member of the cruciferin family was cloned from embryo cDNA and sequenced. The nucleotide sequence of the cruciferin cDNA clone, pC1, contains one long open reading frame, which originates in a hydrophobic signal peptide region. Therefore, the complete sequence of the cruciferin mRNA was obtained by primer extension of the cDNA. The predicted precursor polypeptide is 488 amino acids long, including the 22 amino acids of the putative signal sequence. The amino acid composition of cruciferin protein is very similar to the predicted composition of the precursor. Comparison with an amino acid sequence of legumin from peas, deduced from the nucleotide sequence of a genomic clone, shows that the α polypeptide precedes the β polypeptide on the precursor. Cruciferin and legumin share 40% homology in the regions which can be aligned. However, cruciferin contains a 38 amino acid region high in glutamine and glycine in the middle of the α subunit, which is absent in legumin. Legumin has a highly charged region, 57 amino acids long, at the carboxyl-end of the α subunit, which is not found in cruciferin. Both of these regions appear to have originated by reiteration of sequences. re]19850513 ac]19850715

Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development.

We have analyzed the nucleotide sequence and accumulation of an mRNA which is prevalent in seeds of Brassica napus L. During normal development, the mRNA begins to accumulate during late embryogeny, is stored in dry seeds, and becomes undetectable in seedlings within 24 hours after imbibition. Moreover, abscisic acid treatment of embryos precociously induces or enhances accumulation of the mRNA. Nucleotide sequencing studies show that the deduced 30 kDa polypeptide has an unusual primary structure; the polypeptide possesses direct amino acid sequence repeats and is virtually entirely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Based upon the expression pattern and predicted polypeptide sequence, we conclude that the mRNA is encoded by a late embryogenesis-abundant (Lea) gene in B. napus.

Precociously germinating rapeseed embryos retain characteristics of embryogeny

We compared the germination of Brassica napus L. embryos at three stages of development-mid-cotyledon, maturation and mature dry-to determine at which stage they acquired the capacity for normal germination and seedling development. Embryos were removed from the seed and cultured on hormone-free medium, allowing them to germinate. The transition from embryogeny to germination was monitored both morphologically and biochemically, using synthesis of 12 S storage protein as a marker of embryogeny. The mature embryos (dry seeds) set the standard for normal seedling development: radicle emergence, hypocotyl extension and cotyledon expansion occurred within 2 d and true leaves were formed within a week of germination. Rocket immunoelectrophoresis indicated that the storage proteins in seedlings from mature dry embryos were completely degraded within a week. In contrast, the midcotyledon-stage embryos appeared to germinate abnormally, retaining many embryonic characteristics. Although the roots emerged, the hypocotyls did not elongate and secondary cotyledons instead of leaves were formed at the shoot apex. Also, the seedlings continued to synthesize and accumulate storage proteins. The maturation-stage embryos did develop into normal-looking seedlings, but complete degradation of storage proteins required several weeks, presumably reflecting continued synthesis and turnover. We conclude that embryogenic and germination-specific processes can occur concurrently and that the capacity to develop as normal seedlings is acquired gradually during the maturation process.

Molecular analysis of a cruciferin storage protein gene family of Brassica napus

We have isolated a five-member gene subfamily which encodes cruciferin, a legumin-like 12S storage protein of Brassica napus L., and have analyzed the structure and expression of the family members in developing embryos. Sequence analysis has shown that the coding regions of all five genes are highly similar, with the two most divergent members of the family retaining 89% sequence identity. The analysis of this cruciferin gene familys expression indicates that the developmental pattern of expression of each gene is similar, and the steady-state mRNA levels of each gene are approximately equivalent to each other at all developmental stages.

Differential expression of members of the napin storage protein gene family during embryogenesis inBrassica napus

S1 nuclease analysis and sub-family-specific oligonucleotide probes were used to characterize the expression during embryogenesis of the napin storage protein gene family ofBrassica napus (oilseed rape). The expression of one sub-class represented by the napin gene gNa peaks and declines earlier than the other members of the family. This sub-class was highly expressed representing ca. 20% of napin mRNA at 26 days after anthesis.

Biotechnology is not compatible with sustainable agriculture

Biotechnology increases commercialization of food production, which competes with food for home use. Most people in the world grow their own food, and are more secure without the mediation of the market. To the extent that biotechnology enhances market competitiveness, world food security will decrease. This instability will result in a greater gap between rich and poor, increasing poverty of women and children, less ability and incentive to protect the environment, and greater need for militarization to maintain order. Therefore, biotechnology should be discouraged. An active program to protect and strengthen local food production and to decrease reliance on industrial agriculture should be promoted.

The very structure of scientific research mitigates against developing products to help the environment, the poor, and the hungry

ConclusionsFrom the arguments I have presented, I hope it is clear that the distinction between basic and applied research is tenuous. Certain areas of research and methods may be favoured over others because of intrinsic biases, which are predictive of the type of application possible. Believing in the neutrality of pure knowledge is like wearing blinders: scientists need not be too concerned about the way in which the knowledge they generate is used. In my own case, this belief led to my participation in a system of agriculture to which I do not subscribe. Scientists should be willing to take responsibility for how they function in the system as a whole. Given the social structure of science, it is difficult for basic researchers to see their connections with the larger issues, much less to control them. Perhaps new ways of constructing knowledge-generating societies will be necessary in order to encourage individual responsibility.