The Plant Journal

Gu, Jianying; Dempsey, Sandra; Newton, Kathleen J.

doi: 10.1046/j.1365-313X.1994.6060787.xpmid: 7849753

The maize NCS6 mitochondrial mutation is a partial deletion of the cytochrome oxidase subunit 2 gene (cox2) that survives heteroplasmically in the plant. Mutant mitochondria segregate from normal mitochondria during somatic development giving rise to defective sectors on the plants, including areas of kernel abortion on the ears. Embryos from NCS6 kernels can be rescued by tissue culture. Slowly growing Type II callus derived from one of these embryos has been shown by PCR analysis to be homoplasmic for the mutation, carrying only the defective mitochondrial cox2 gene. Most of the rescued embryos were heteroplasmic for normal and mutant genes and heteroplasmy was maintained in the callus cultures. However, when suspension cultures were initiated from heteroplasmic calli, normal cells were shown to have a selective advantage. When the homoplasmic cox2 mutant callus cultures were placed on regeneration medium, plantlets did not regenerate. Heteroplasmic calli were capable of regeneration under the same conditions. These studies suggest that the functioning of mitochondrial cytochrome oxidase is not essential for growth as callus, but is required for the differentiation and development of plants.

Kang, Fan; Rawsthorne, Stephen

doi: 10.1046/j.1365-313X.1994.6060795.xpmid: N/A

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP‐glucose‐6‐phosphate dehydrogenase, NADP‐6‐phosphogluconate dehydrogenase, fructose‐1,6‐bisphosphatase, NADP‐malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl‐CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with 14C‐labelled carbon precursors and the incorporation of 14C into starch and fatty acids was determined. 14C from glucose‐6‐phosphate (G‐6‐P), fructose, glucose, fructose‐6‐phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness‐ and ATP‐dependent manner. The rate of starch synthesis was highest from G‐6‐P, although fructose gave rates which were 70% of those from G‐6‐P. Glucose‐1‐phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G‐6‐P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G‐6‐P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.

Kumar, Vinod; Trick, Martin

doi: 10.1046/j.1365-313X.1994.6060807.xpmid: 7849754

The S‐receptor kinase (SRK) gene which is implicated in the self‐incompatibility system of Brassica oleracea is one member of a large and complex family of similar sequences. Genomic and cDNA clones were isolated for the authentic, S‐linked SRK29 gene and its DNA sequence determined. Reverse transcriptase PCR (RT‐PCR) was used to detect the expression of SRK,29 and other members of the family in stigma, leaf and root tissues. The SRK was found to be stigma‐specific whereas, for instance, K3 transcripts appeared in all three tissues. The RT‐PCR analysis also demonstrated the existence of partially processed intermediates for several of the kinase transcripts and, in the case of SRK29, a product apparently resulting from the splicing of a cryptic intron. RFLP analysis of an F2 family segregating for the S29 allele was used to show S‐linkage for the SRK and possibly for the K2 sequence. The K8 kinase probe also revealed a minor RFLP which segregated with the S‐locus.

Chaudhry, Bushra; Müller‐Uri, Frieder; Cameron‐Mills, Verena; Gough, Simon; Simpson, David; Skriver, Karen; Mundy, John

doi: 10.1046/j.1365-313X.1994.6060815.xpmid: 7849755

The N‐terminal region of a 60 kDa, jasmonate‐induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome‐inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N‐terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome‐inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.

Albà, M. Mar; Culiáñez‐Macià, Francisco A.; Goday, Adela; Freire, Miguel Angel; Nadal, Belén; Pagès, Montserrat

doi: 10.1046/j.1365-313X.1994.6060825.xpmid: N/A

A developmentally and environmentally regulated gene in maize, MA16, encoding an RNA‐binding protein that binds preferentially to uridine and guanosine‐rich RNAs has previously been described. To gain some insight into the function of MA16 the distribution of MA16 mRNA and protein during maize development was investigated using in situ hybridization, RNA and protein gel blot analysis and immunocytochemistry. The results show that MA16 is expressed throughout development of the embryo and seedling in different tissues and at different levels. The level of MA16 mRNA is higher in developing and expanding structures such as the root elongation zone and young leaves. After stress treatment MA16 mRNA increases in total and polysomal RNA, but no significant change in the level of the protein was detected. MA16 is a non‐ribosomal nucleolar protein. Using immunoelectron microscopy the MA16 protein has been located in the dense fibrillar component and to a lesser extent in the granular component of the nucleolus. It was found that MA16 contains the conserved sequence motifs R(G)nY(G)nR and RR(E/D)(G)nY(G)n repeated in the C‐terminal of the molecule that conforms imperfectly to the GAR motif proposed for nucleolar proteins. In light of these results the stress regulation of MA16 and a likely role for this protein in pre‐rRNA processing and/or ribosome assembly is discussed.

Callos, Joseph D.; DiRado, Marion; Xu, Bibo; Behringer, Friedrich J.; Link, Bruce M.; Medford, June I.

doi: 10.1046/j.1365-313X.1994.6060835.xpmid: 7849756

In plant development, leaf primordia are formed on the flanks of the shoot apical meristem in a highly predictable pattern. The cells that give rise to a primordium are sequestered from the apical meristem. Maintenance of the meristem requires that these cells be replaced by the addition of new cells. Despite the central role of these activities in development, the mechanism controlling and coordinating them is poorly understood. These processes have been characterized in the Arabidopsis mutant forever young (fey). The fey mutation results in a disruption of leaf positioning and meristem maintenance. The predicted FEY protein shares significant homology to a nodulin and limited homology to various reductases. It is proposed that FEY plays a role in communication in the shoot apex through the modification of a factor regulating meristem development.

Kalla, Roger; Shimamoto, Ko; Potter, Robert; Nielsen, Peter Stein; Linnestad, Casper; Olsen, Odd‐Arne

doi: 10.1046/j.1365-313X.1994.6060849.xpmid: 7849757

This paper describes the aleurone‐specific gene Ltp2 from barley, which encodes a putative 7 kDa non‐specific lipid transfer protein. As shown by Northern and in situ hybridization analyses, the Ltp2 transcript is present in barley aleurone cells shortly after the initiation of aleurone cell differentiation. The expression of Ltp2 increases until grain mid‐maturity, but the mRNA is absent from mature grains. The Ltp2 transcript is undetectable in the embryo and vegetative tissues, confirming the aleurone specificity of the Ltp2 gene. The ability of the isolated 801 bp Ltp2 promoter to direct aleurone‐specific expression in immature barley grains is demonstrated by particle bombardment experiments. In these experiments, the activity of the Ltp2 promoter is 5% of the activity of the strong constitutive Actin1 promoter from rice, as quantified by GUS activity measurements. In stably transformed rice plants containing the Ltp2 promoter—Gus construct, the specificity of the Ltp2 promoter is confirmed in vivo by the presence of GUS activity exclusively in the aleurone layer. This study demonstrates the conserved nature of the regulatory signals involved in aleurone‐specific gene transcription in cereal grains.

Blokland, R.; Geest, N.; Mol, J.N.M.; Kooter, J.M.

doi: 10.1046/j.1365-313X.1994.6060861.xpmid: N/A

Co‐suppression of the pigmentation gene chalcone synthase (chs) in Petunia hybrida by chs transgenes leads to white or variegated flowers and is characterized by a reduction in steady‐state mRNA levels. To determine the level at which suppression occurs different petunia transformants were analysed containing CaMV‐35S RNA promoter‐driven hybrid genes consisting of the β‐glucuronidase gene (uidA) linked to the full‐length chsA cDNA, the 5′ half or to the 3′ half. With these transgenes one out of 12–15 primary transformants showed suppression of the transgenes and of the resident chs genes throughout the flower or in sectors. The reduction in steady‐state chs mRNA was not the result of a transcriptional inactivation event. As determined by nuclear run‐on experiments, transcription of suppressed chs genes was similar to that of non‐suppressed genes. This indicates that co‐suppression occurs post‐transcriptionally. Among individual transformants the transgenes were transcribed at different levels but neither a high nor a low level correlated with a particular degree or pattern of suppression. Surprisingly, even a promoterless chs transgene construct was found to suppress the endogenous chs genes in three out of 15 transformants. It remains, however, unknown whether or not transcription of the transgene locus is required to induce co‐suppression. The data suggest that properties of the chs transgene locus other than the expression level are important for inducing co‐suppression. The possible role of antisense RNA, which was detected in all transformants, ectopic pairing and the structure of the integrated T‐DNAs in the mechanism of the selective increase in chs RNA turnover are discussed.

Hewelt, Andreas; Prinsen, Els; Schell, Jeff; Onckelen, Harry; Schmülling, Thomas

doi: 10.1046/j.1365-313X.1994.6060879.xpmid: 7849758

Tobacco plants have been transformed with a T‐DNA construct harboring a promoterless cytokinin‐synthesizing ipt gene close to the right T‐DNA border. Eighteen out of 85 transgenic clones displayed phenotypic alterations typical for an enhanced cytokinin production. Northern blot analysis confirmed the transcriptional activation of the introduced gene by tagged plant promoters. The concentration of cytokinins, expressed as zeatinriboside equivalents, was increased up to sevenfold in transgenic tissues. These increases in cytokinin levels resulted in major developmental changes. Transgenic clones exhibited to different levels traits of a general cytokinin‐syndrome, i.e. reduced root growth, reduced apical dominance, reduced leaf surface, reduced growth of the stem and retarded leaf senescence or displayed localized and developmentally specific cytokinin‐induced alterations in otherwise normally developing plants. These traits were in particular a simultaneous break of dormancy in all axillary buds before or at the onset of flowering or the reorientation of the developmental pathway of secondary meristems or terminally differentiated cells. This indicates that endogenously produced cytokinins not only influence different growth parameters but have the potential to alter differentiation pattern. The results show that stably inherited developmental alterations due to a general or localized cytokinin overproduction can be obtained by the promoter‐tagging approach. The investigation of gene dosage effects in homozygote plants readresses the question of threshold levels for cytokinin effects on the developmental program of plants.

Graham, Ian A.; Baker, Christopher J.; Leaver, Christopher J.

doi: 10.1046/j.1365-313X.1994.6060893.xpmid: 7849759

Recently it has been demonstrated that the single‐copy malate synthase (MS) and isocitrate lyase (ICL) genes from cucumber are regulated by nutritional status in cucumber cell cultures. In this paper a new cucumber mesophyll protoplast transient expression system is described in which electroporated MS promoter—GUS reporter gene constructs exhibit the same pattern of expression as the endogenous MS gene. Both the electroporated MS—GUS constructs and the endogenous gene are expressed when protoplasts are cultured for 48 h on a non‐metabolizable carbon source such as mannitol or 3‐methylglucose, and repressed when cultured on a utilizable carbon source such as sucrose, glucose or fructose. A series of deletion mutants identified a region from position −248 to −125 relative to the start of transcription that is essential for expression of the MS—GUS construct under the different metabolic conditions. A 191 bp fragment spanning this region was fused, in both orientations, to the CaMV 35S core promoter. A pattern of metabolic regulation similar to that of the intact MS promoter was observed for these promoter fusion constructs which strongly suggests the presence of enhancer element(s) within this region. Comparison of the 191 bp region with other MS and ICL promoter sequences revealed a region of homology, designated RT. A gel retardation assay was used to assess binding of the 191 bp fragment to whole cell protein extracts from cell cultures expressing MS. Both the unlabelled 191 bp fragment and a synthetic oligonucleotide of RT compete specifically for the demonstrated binding activity.