I. Marta Evans

The POLARIS Gene of Arabidopsis Encodes a Predicted Peptide Required for Correct Root Growth and Leaf Vascular Patterning

The POLARIS (PLS) gene of Arabidopsis was identified as a promoter trap transgenic line, showing β-glucuronidase fusion gene expression predominantly in the embryonic and seedling root, with low expression in aerial parts. Cloning of the PLS locus revealed that the promoter trap T-DNA had inserted into a short open reading frame (ORF). Rapid amplification of cDNA ends PCR, RNA gel blot analysis, and RNase protection assays showed that the PLS ORF is located within a short (∼500 nucleotides) auxin-inducible transcript and encodes a predicted polypeptide of 36 amino acid residues. pls mutants exhibit a short-root phenotype and reduced vascularization of leaves. pls roots are hyperresponsive to exogenous cytokinins and show increased expression of the cytokinin-inducible gene ARR5/IBC6 compared with the wild type. pls seedlings also are less responsive to the growth-inhibitory effects of exogenous auxin and show reduced expression of the auxin-inducible gene IAA1 compared with the wild type. The PLS peptide-encoding region of the cDNA partially complements the pls mutation and requires the PLS ORF ATG for activity, demonstrating the functionality of the peptide-encoding ORF. Ectopic expression of the PLS ORF reduces root growth inhibition by exogenous cytokinins and increases leaf vascularization. We propose that PLS is required for correct auxin-cytokinin homeostasis to modulate root growth and leaf vascular patterning.

Characterisation of the storage protein subunits synthesised in vitro by polyribosomes and RNA from developing pea (Pisum sativum L.) : II. Vicilin.

Evidence is presented to show that legumin, the major storage protein in Pisum, is synthesised in vitro by the wheat germ and reticulocyte lysate systems, from polyribosomes and mRNA isolated from developing pea seeds. While legumin isolated from mature pea seeds consists of 40,000 and 20,000 MW subunits, the in vitro legumin is synthesised as a 60,000 MW precursor consisting of covalently linked 40,000 and 20,000 MW subunits. The implications of these findings are discussed in relationship to studies with other systems.

A gene from pea (Pisum sativum L.) with homology to metallothionein genes

While searching for ‘organ‐specific’ genes in pea (Pisum sativum L.) we have isolated a gene (designated PsMT A) which has an ORF encoding a predicted protein with some similarity to metallothioneins (MTs). The PsMT A transcript is abundant in roots which have not been exposed to elevated concentrations of trace metals.

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.)

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.

Regulation of the transcription of storage-protein mRNA in nuclei isolated from developing pea (Pisum sativum L.) cotyledons.

Two types of storage protein, vicillin and legumin, occur in the developing pea seed. Storage-protein gene expression has been studied during cotyledon development by assaying specific transcripts produced by nuclei isolated at different stages, and from pea leaves. The proportion of vicilin to legumin transcripts changed during development: vicilin transcripts predominated at 9 and 11 days after flower opening (d.a.f.) and were similar in amount to legumin at 14 d.a.f., whereas at 18 d.a.f., legumin transcripts predominated and little vicilin transcription was observed. The rate of storage-protein transcription correlated with previously determined (Gatehouse et al. 1982) mRNA levels during seed development; these transcripts were not detected in similar assays using leaf nuclei. Transcription by cotyledonary nuclei for short times indicated that post-transcriptional processing may be a factor in regulating mRNA levels, at least in the earlier part of seed development.

Expression of a Brassica napus extensin gene in the vascular system of transgenic tobacco and rape plants

The organs and tissues where the Brassica napus extA extensin gene is expressed have been identified. The extA gene with 3.75 kb of 5′ flanking sequence was transferred to tobacco via disarmed Agrobacterium tumefaciens vectors and transgenic plants regenerated. The gene was found to be inactive in transgenic tobacco leaf, but was active as measured by RNA transcript assays in both stem and root tissues. To determine the cell-specific expression pattern of the extA gene, a promoter-reporter gene fusion construct was made consisting of 1.0 kb of 5′ extA sequence fused to the coding region of the glucuronidase (GUS) gene. This fusion construct was introduced into B. napus via Agrobacterium rhizogenes, and expression of GUS in transgenic rape hairy roots was examined. GUS activity was only seen in the vascular tissues of the rape root, and was found to be specifically localised in the phloem.

Characterisation of cDNA and genomic clones encoding homologues of the 65 kDa regulatory subunit of protein phosphatase 2A in Arabidopsis thaliana

Two cDNA species encoding sequences homologous to the 65 kDa regulatory subunit (PR 65) of protein phosphatase 2A (PP2A) have been isolated from an Arabidopsis thaliana cDNA library. These were designated pDF1 and pDF2. pDF1 is 1795 bp long and by comparison with the human and porcine PP2A regulatory subunit sequences represents a full-length clone. It encodes a predicted polypeptide of 587 amino acid residues. pDF2 is truncated at the 5′ end by 237 bp. The complete nucleotide sequences have been determined for both cDNA species. Comparison of the nucleotide and the deduced amino acid sequences showed that the two sequences were homologous but not identical and therefore must be derived from two different genes. Northern blot analysis was performed on total RNA and poly(A)+ RNA isolated from seed at various stages of development and from young leaf material of Brassica napus L. (oilseed rape). Both cDNA probes hybridised to a single major mRNA species of ca. 2.2 kb. The highest level of expression was observed in the total RNA from developing rape seed at about 33 days after flowering, and the transcript level in the poly(A)+ RNA of the seed was higher than in young leaf of oilseed rape. Southern blot analysis was performed on two varieties of A. thaliana and B. napus genomic DNA; this identified a small family of genes in A. thaliana consisting of at least 2 or 3 members and a larger multigene family in B. napus of at least 5 or 6 members.Two independent genomic clones were isolated from an A. thaliana genomic library. Sequencing of a fragment common to both revealed that the sequence was identical in both clones and, therefore, they were assumed to contain the same genomic sequence. The genomic sequence selected, designated regA, is 3639 bp long and the coding sequence contains eleven introns. The gene encodes a predicted polypeptide of 590 amino acid residues. The sequence comparison with both cDNA sequences showed that it is homologous but not identical to the two, confirming that at least three different genes exist in A. thaliana which encode PR65 of PP2A.

Transcriptional and posttranscriptional regulation of seed storage-protein gene expression in pea (Pisum sativum L.).

At least three classes of legumin, encoded by the gene families legA, legJ and legS, and a lectin, encoded by a single gene, accumulate in the developing cotyledons of Pisum sativum L. Transcription rates for the genes encoding these proteins were measured in nuclei isolated from cotyledons at 12 and 16 days after flowering (DAF). The steady-state levels of the corresponding mRNA species were also measured in absolute terms throughout cotyledon development, from 8–9 to 28 DAF. When transcription rates and steady-state mRNA levels of the different gene families are compared, there is little correlation. This indicates a posttranscriptional regulation of the level of expression of these storage proteins in the developing cotyledons. Expression of the legumin genes is known to be seed-specific, whereas expression of the lectin gene is found in both seed and root. When transcription rates were measured in leaf nuclei the levels of legumin and lectin transcripts detected approached background levels, indicating that these genes are either inactive or transcribed at very low levels in leaves; however, the rate of transcription of the chlorophyll a/b-binding protein gene was high. This points to transcriptional control as the major factor in the organ-specificity of legumin and lectin expression.

Genes with similarity to metallothionein genes and copper, zinc ligands in Pisum sativum L.

The PsMT gene family of pea (Pisum sativum L.) encodes predicted proteins with sequence similarity to metallothioneins. However, PsMT proteins have not yet been characterised in planta and their functions remain obscure. PsMT transcripts were identified in the cortex tissue of pea roots using tissue squash-blotting techniques. Transcripts were not detected on northern blots of RNA isolated from the embryonic radicle, but PsMT transcript abundance in roots increased with age of germinating seedlings. The PsMT A gene was expressed in E. coli as a carboxyterminal extension of glutathione-S-transferase (GST). Fusion protein purified from crude cell lysates (500 mL cultures) bound an estimated amount of 5.99, 6.27 and 7.07 moles of Zn, Cu and Cd respectively per mole protein, compared to equivalent estimates of 0.37, 0.63 and 0.26 moles for GST alone. Similar estimates for Fe-binding were 0.28 moles for GST-PsMT A fusion protein and 0.1 moles for GST alone.

Proteins of some legumes with reference to environmental factors and nutritional value

Storage globulins contribute 80–90% of the seed protein of many legumes and consist principally of two proteins called vicilin and legumin in peas. The relative proportions of the two proteins has been shown to differ in different legumes by gel-electrophoresis. The essential amino acid composition of vicilin and legumin ofPisum andVicia differ and the possible significance of these findings to screening strategies for improved legume programmes was discussed. Cowpeas were grown in growth cabinets under standard environmental conditions. Seeds were shown to have the same percentage nitrogen and sulphur on a dry-weight basis, irrespective of the position of the pod on the plant, indicating that legume seeds may have a conservative biochemistry.