Degrading chloroplast mRNA: the role of polyadenylation.

Chloroplast development involves changes in the stability of specific plastid mRNAs. To understand how the half-lives of these mRNAs are modified, several laboratories are investigating how plastid mRNAs are degraded. This has led to the isolation of a high-molecular-weight complex that contains an endoribonuclease and a 3'-5' exoribonuclease, and the discovery that efficient mRNA degradation requires polyadenylation. These findings are similar to recent discoveries in Escherichia coli. However, an important difference between the two systems is that chloroplast mRNA degradation involves nuclear-encoded proteins. Modification of these proteins could provide the mechanism for altering plastid-mRNA half-lives in response to developmental stimuli.

Changes in Chloroplast mRNA Stability during Leaf Development.

During spinach leaf development, chloroplast-encoded mRNAs accumulate to different steady-state levels. Their relative transcription rates alone, however, cannot account for the changes in mRNA amount. In this study, we examined the importance of mRNA stability for the regulation of plastid mRNA accumulation using an in vivo system to measure mRNA decay in intact leaves by inhibiting transcription with actinomycin D. Decay of psbA and rbcL mRNAs was assayed in young and mature leaves. The psbA mRNA half-life was increased more than twofold in mature leaves compared with young leaves, whereas rbcL mRNA decayed with a similar relative half-life at both leaf developmental stages. The direct in vivo measurements demonstrated that differential mRNA stability in higher plant plastids can account for differences in mRNA accumulation during leaf development. The role of polysome association in mRNA decay was also investigated. Using organelle-specific translation inhibitors that force mRNAs into a polysome-bound state or deplete mRNAs of ribosomes, we measured mRNA decay in vivo in either state. The results showed that rbcL and psbA mRNAs are less stable when bound to polysomes relative to the polysome-depleted mRNAs and that their stabilities are differentially affected by binding to polysomes. The results suggested that ribosome binding and/or translation of the psbA and rbcL mRNAs may function to modulate the rate of their decay in chloroplasts.

Plant farnesyltransferase can restore yeast Ras signaling and mating.

Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes.

RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein.

Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein. We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3' untranslated regions.

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3' untranslated region (3'UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3'UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3'UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca(2+)-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3'-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (B(max)) 0.25 time for petD 3'UTR, and three times for psbA 3'UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that B(max), not K(d), might be a more important factor in the P-24RNP-3'UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.

Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif.

Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity.

Arachidonic acid alters tomato HMG expression and fruit growth and induces 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent lycopene accumulation

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.

Empowering biotechnology in southern Africa: establishment of a robust transformation platform for the production of transgenic industry-preferred cassava.

Knowledge and technology transfer to African laboratories and farmers is an important objective for achieving food security and sustainable crop production on the sub-Saharan African continent. Cassava (Manihot esculenta Crantz) is a vital source of calories for more than a billion people in developing countries, and its potential industrial use for starch and bioethanol in the tropics is increasingly being recognized. However, cassava production remains constrained by the susceptibility of the crop to several biotic and abiotic stresses. For more than a decade, biotechnology has been considered an attractive tool to improve cassava as it substantially circumvents the limitations of traditional breeding, which is particularly time-consuming and tedious because of the high heterozygosity of the crop. A major constraint to the development of biotechnological approaches for cassava improvement has been the lack of an efficient and robust transformation and regeneration system. Despite some success achieved in genetic modification of the model cassava cultivar Tropical Manihot Series (TMS), TMS 60444, in some European and U.S. laboratories, the lack of a reproducible and robust protocol has not allowed the establishment of a routine transformation system in sub-Saharan Africa. In this study, we optimized a robust and efficient protocol developed at ETH Zurich to successfully establish transformation of a commercially cultivated South African landrace, T200, and compared this with the benchmark model cultivar TMS 60444. Results from our study demonstrated high transformation rates for both T200 (23 transgenic lines from 100 friable embryogenic callus (FEC) clusters) compared with TMS 60444 (32 transgenic lines from 100 FEC clusters). The success in transforming landraces or farmer-preferred cultivars has been limited, and the high transformation rate of an industry-preferred landrace in this study is encouraging for a feasible transformation program for cassava improvement in South Africa (SA), which can potentially be extended to other countries in southern Africa. The successful establishment of a robust cassava transformation and regeneration system in SA demonstrates the relevance of technology transfer to sub-Saharan Africa and highlights the importance of developing suitable and reliable techniques before their transfer to laboratories offering less optimal conditions.

Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.

Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.

Biofortification of essential nutritional compounds and trace elements in rice and cassava.

Plant biotechnology can make important contributions to food security and nutritional improvement. For example, the development of 'Golden Rice' by Professor Ingo Potrykus was a milestone in the application of gene technology to deliver both increased nutritional qualities and health improvement to wide sections of the human population. Mineral nutrient and protein deficiency as well as food security remain the most important challenges for developing countries. Current projects are addressing these issues in two major staple crops, cassava (Manihot esculenta Crantz) and rice. The tropical root crop cassava is a major source of food for approximately 600 million of the population worldwide. In sub-Saharan Africa >200 million of the population rely on cassava as their major source of dietary energy. The nutritional quality of the cassava root is not sufficient to meet all dietary needs. Rice is the staple food for half the world population, providing approximately 20% of the per capita energy and 13% of the protein for human consumption worldwide. In many developing countries the dietary contributions of rice are substantially greater (29.3% dietary energy and 29.1% dietary protein). The current six most popular 'mega' rice varieties (in terms of popularity and acreage), including Chinese hybrid rice, have an incomplete amino acid profile and contain limited amounts of essential micronutrients. Rice lines with improved Fe contents have been developed using genes that have functions in Fe absorption, translocation and accumulation in the plant, as well as improved Fe bioavailability in the human intestine. Current developments in biotechnology-assisted plant improvement are reviewed and the potential of the technology in addressing human nutrition and health are discussed.

Differences in pattern of a DNA protein complex isolated from vegetative cells and spores of Bacillus subtilis.

A DNA protein complex has been isolated from vegetative cells and spores of Bacillus subtilis. Properties of the DNA protein complex prepared from vegetative cells were studied and SDS gel electrophoresis was employed to compare the different DNase-untreated and -treated DNA protein complexes. It is concluded that proteins are associated with the DNA and differences in protein pattern in polyacrylamide gels indicates the involvement of DNA-binding proteins in the regulation of spore formation.

A 43 kD light-regulated chloroplast RNA-binding protein interacts with the psbA 5' non-translated leader RNA.

Expression of the chloroplast psbA gene coding for the D1 protein of Photosystem II is subject to regulation at different levels in higher plants, including control of mRNA accumulation and translation. In dicots, the conserved 5' non-translated leader (5'-UTR) of the psbA mRNA is sufficient to direct the light-dependent translation of the D1 protein. In this report we show that the psbA mRNA 5'-UTR forms a stem-loop structure and binds a 43 kD chloroplast protein (43RNP). Binding of the 43RNP is sensitive to competition with poly(U), but insensitive to high concentrations of tRNA, the RNA homopolymers poly(A), poly(G), poly(C), or poly(A):poly(U) as a double-strand RNA. The 43RNP does not bind efficiently to the psbA mRNA 3' non-translated region, although the RNA sequence is U-rich and folds into a stem-loop. A deletion mutant of the psbA 5'-UTR RNA in which 5' sequences of the stem-loop are removed does not affect 43RNP binding. Together, these properties suggest that the 43RNP binds most effectively to a specific single-strand U-rich sequence preceding the AUG start codon in the psbA mRNA. Binding of the 43RNP is not detectable in plastid protein extracts from 5-day-old dark-grown seedlings, but is detectable in light-grown seedlings as well as mature plants in the light and after shifted to the dark. The 43RNP is therefore a candidate for a regulatory RNA-binding protein that may control the accumulation and/or translation of the psbA mRNA during light-dependent seedling development.

Chloroplast mRNA 3' end processing requires a nuclear-encoded RNA-binding protein.

The protein coding regions of plastid mRNAs in higher plants are generally flanked by 3' inverted repeat sequences. In spinach chloroplast mRNAs, these inverted repeat sequences can fold into stem-loop structures and serve as signals for the correct processing of the mature mRNA 3' ends. The inverted repeat sequences are also required to stabilize 5' upstream mRNA segments, and interact with chloroplast protein in vitro. To dissect the molecular components involved in chloroplast mRNA 3' end processing and stability, a spinach chloroplast protein extract containing mRNA 3' end processing activity was fractionated by FPLC and RNA affinity chromatography. The purified fraction consisted of several proteins and was capable of processing the 3' ends of the psbA, rbcL, petD and rps14 mRNAs. This protein fraction was enriched for a 28 kd RNA-binding protein (28RNP) which interacts with both the precursor and mature 3' ends of the four mRNAs. Using specific antibodies to this protein, the poly(A) RNA-derived cDNA for the 28RNP was cloned and sequenced. The predicted amino acid sequence for the 28RNP reveals two conserved RNA-binding domains, including the consensus sequences RNP-CS1 and CS2, and a novel acidic and glycine-rich N-terminal domain. The accumulation of the nuclear-encoded 28RNP mRNA and protein are developmentally regulated in spinach cotyledons, leaves, root and stem, and are enhanced during light-dependent chloroplast development. The general correlation between accumulation of the 28RNP and plastid mRNA during development, together with the result that depletion of the 28RNP from the chloroplast protein extract interferes with the correct 3' end processing of several chloroplast mRNAs, suggests that the 28RNP is required for plastid mRNA 3' end processing and/or stability.

Developmental, organ-specific, and light-dependent expression of the tomato ribulose-1,5-bisphosphate carboxylase small subunit gene family.

The tomato gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] has five genes, designated Rbcs-1, -2, -3A, -3B, and -3C. We have measured the steady-state mRNA levels for each of the five genes in various tomato organs using gene-specific oligonucleotides. All five genes are highly expressed in leaves, and transcripts of two genes, Rbcs-3B and Rbcs-3C, account for approximately equal to 60% of the total leaf transcripts. The relative transcript levels in the stem, in nature fruits, and etiolated seedlings (plants germinated and grown in the dark) correspond to 3.2%, 6.5%, and 4.6%, respectively, of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA level in leaves, and no transcripts have been detected in roots and ripe tomato fruits. Only Rbcs-1 and Rbcs-2 are expressed during the photosynthetically active phase of fruit development. Transcripts from these genes and from the Rbcs-3A locus are also present in etiolated seedlings. Rbcs-3B and Rbcs-3C transcripts, which are the most abundant mRNAs of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family in the leaf, are undetectable in dark-grown seedlings and immature fruit. The selective expression of Rbcs-1 and Rbcs-2 in the dark and in the pericarp of green fruit and the induction and rapid mRNA accumulation for Rbcs-3B and Rbcs-3C after illumination may reflect different regulatory mechanism(s) that control the expression of individual members in the tomato ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family.

Diurnal mRNA fluctuations of nuclear and plastid genes in developing tomato fruits.

Steady-state transcript levels of nuclear (rbcS, cab) and plastid (rbcL, psbA) encoded photosynthesis-specific genes were determined at noon and 05.00 h in different developmental stages of tomato fruits (7-35 days after anthesis). Small alterations are observed in mRNA levels for the small subunit (ssu) and large subunit (lsu) of RuBPC/Oase and the QB-binding protein of photosystem II at these two time-points, while significant steady-state transcript level fluctuations are detectable for the light harvesting complex protein. LHCP II transcripts accumulate during the day, and decline to low levels during the night. In contrast, the LHC II protein levels remain constant during the same period of development. A detailed analysis of transcript levels of the nuclear and plastid genes at 4-h intervals throughout a 38-h period demonstrates that LHCP II mRNA accumulation is highest at noon and lowest at 04.00 h. The transcripts of the ssu and lsu of RuBPC/Oase, photosystem I and II reaction center proteins, as well as the beta-subunit of the mitochondrial ATPase and the beta-subunit of tubulin, accumulate during the night and decrease to low levels in the afternoon. The transcript levels of the genes examined in this study fluctuate with certain periodicities. We suggest that gene expression in developing tomato fruits is at least partially controlled by diurnal rhythms, which are therefore also operational in other organs, besides leaves, of higher plants.

Analysis of promoter regions for the spinach chloroplast rbcL, atpB and psbA genes.

A promoter-deletion derivative of the spinach trnM2 gene was used for the identification and characterization of the promoter regions for the spinach chloroplast RuBisCo large subunit (rbcL), ATPase beta-subunit (atpB) and QB-polypeptide (psbA) genes. The DNA sequences 5' upstream from the transcriptional start sites of these genes share homology with the ctp1 and ctp2 arrangement found for the trnM2 transcription unit and the canonical Escherichia coli '-10' and '-35' promoter regions. Synthetic DNA fragments of approximately 40-bp regions, including the defined transcriptional start sites and proximal residues, from rbcL, atpB and psbA, were fused to the trnM2 deletion mutant 51. The promoter-fusion constructs direct the correct transcription of tRNAMet2 in the chloroplast extract with distinct efficiencies. The ctp1- and ctp2-like elements in the trnM2, rbcL and psbA promoter regions can be interchanged to yield functional chimeric promoters of varying strengths. As a result, ctp1 sequences from atpB and psbA, trnM2 and rbcL, respectively, can be ordered TTGACA greater than TTGCTT greater than TTGCGC with respect to their intrinsic strengths. Single base pair changes were introduced into the ctp2-like element in the psbA promoter region. In analogy to similar base pair changes which lower promoter efficiency in E. coli, these mutations result in reduced transcription levels in the chloroplast extract. The data are consistent with a prokaryotic model for chloroplast promoter function.