Improving water use in crop production.

Globally, agriculture accounts for 80-90% of all freshwater used by humans, and most of that is in crop production. In many areas, this water use is unsustainable; water supplies are also under pressure from other users and are being affected by climate change. Much effort is being made to reduce water use by crops and produce 'more crop per drop'. This paper examines water use by crops, taking particularly a physiological viewpoint, examining the underlying relationships between carbon uptake, growth and water loss. Key examples of recent progress in both assessing and improving crop water productivity are described. It is clear that improvements in both agronomic and physiological understanding have led to recent increases in water productivity in some crops. We believe that there is substantial potential for further improvements owing to the progress in understanding the physiological responses of plants to water supply, and there is considerable promise within the latest molecular genetic approaches, if linked to the appropriate environmental physiology. We conclude that the interactions between plant and environment require a team approach looking across the disciplines from genes to plants to crops in their particular environments to deliver improved water productivity and contribute to sustainability.

Computational studies of Family A and Family B GPCRs.

A full picture of the similarities between Family A and Family B GPCRs (G-protein coupled receptors) has been frustrated by the lack of clear homology between the respective sequences. Here, we review previous computational studies on GPCR dimerization in which the putative dimerization interfaces have been analysed using entropy, the ET (evolutionary trace) method and related methods. The results derived from multiple sequence alignments of Family A subfamilies have been mapped on to the rhodopsin crystal structure using standard alignments. Similarly, the results for the Family B alignments have been mapped on to the rhodopsin crystal structure using the 'cold-spot' alignment. For both Family A and Family B GPCRs, the sequence analysis indicates that there are functional sites on essentially all transmembrane helices, consistent with the parallel daisy chain model of GPCR oligomerization in which each GPCR makes interactions with a number of neighbouring GPCRs. The results are not too sensitive to the quality of the alignment. Molecular Dynamics simulations of the activation process within a single transmembrane bundle of the rhodopsin and the beta(2)-adrenergic receptor have been reviewed; the key observation, which is consistent with other computational studies, is that there is a translation and bending of helix 6, which contributes to a significant opening out of the intracellular face of the receptor, as shown in the accompanying movies. The simulations required the application of specific experiment-derived harmonic and half-harmonic distance restraints and so the application of such simulations to Family B GPCRs requires considerable care because of the alignment problem. Thus, in order to address the alignment problem, we have exploited the observation that GCR1, a plant GPCR, has homology with Family A, Family B and Family E GPCRs. The resulting alignment for transmembrane helix 3 is presented.

Transient expression of Arabidopsis thaliana ascorbate peroxidase 3 in Nicotiana benthamiana plants infected with recombinant potato virus X.

We have explored the transient over-expression of Arabidopsis thaliana ascorbate peroxidase 3 (APX3) in Nicotiana benthamiana using a viral vector based on the potato virus X (PVX). Plants infected with a PVX:APX3 hybrid had a similar progression of viral particles compared to control plants infected with a PVX:GFP hybrid, indicating that infection was not affected by the over-expression of heterologous APX3. Our results also showed that in PVX:APX3-infected plants, the hybrid virus directed a high level of APX3 expression and the recombinant protein was functional, as inferred from the higher APX activity compared to mock and PVX:GFP hybrid-infected plants. The PVX recombinant expression system used is a simple and quick method for transient expression of heterologous APXs, which are expected to suffer specific processing in plant cells.

Characterisation of pea cytosolic glutathione reductase expressed in transgenic tobacco.

Expression in transgenic tobacco (Nicotiana tabacum L.) of a pea (Pisum sativum L.) GOR2 cDNA, encoding an isoform of glutathione reductase (GOR2), resulted in a 3- to 7-fold elevation of total foliar glutathione reductase (GR) activity. The enzyme encoded by GOR2 was confirmed to be extraplastidial in organelle fractionation studies and was considered most likely to be localised in the cytosol. A partial purification of GOR2 was achieved but a standard affinity chromatography step, using adenosine-2',5'-diphosphate-Sepharose and often employed in the purification of GR from diverse sources, was unsuccessful with this isoform. Preparative isoelectric focussing was employed as part of the purification procedure of GOR2 and a complete separation from plastidial/mitochondrial glutathione reductase (GOR1) was achieved. The isoform GOR2 was shown to have a slower migration on non-denaturing polyacrylamide gels compared with GOR1 and properties typical of GR enzymes from plant sources.

pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation.

Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures. pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an Internet site (http://www.pgreen.ac.uk) through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.

Post-transcriptional regulation prevents accumulation of glutathione reductase protein and activity in the bundle sheath cells of maize.

Glutathione reductase (GR; EC 1.6.4.2) activity was assayed in bundle sheath and mesophyll cells of maize (Zea mays L. var H99) from plants grown at 20 degrees C, 18 degrees C, and 15 degrees C. The purity of each fraction was determined by measuring the associated activity of the compartment-specific marker enzymes, Rubisco and phosphoenolpyruvate carboxylase, respectively. GR activity and the abundance of GR protein and mRNA increased in plants grown at 15 degrees C and 18 degrees C compared with those grown at 20 degrees C. In all cases GR activity was found only in mesophyll fractions of the leaves, with no GR activity being detectable in bundle sheath extracts. Immunogold labeling with GR-specific antibodies showed that the GR protein was exclusively localized in the mesophyll cells of leaves at all growth temperatures, whereas GR transcripts (as determined by in situ hybridization techniques) were observed in both cell types. These results indicate that post-transcriptional regulation prevents GR accumulation in the bundle sheath cells of maize leaves. The resulting limitation on the capacity for regeneration of reduced glutathione in this compartment may contribute to the extreme chilling sensitivity of maize leaves.

Plant cell-directed control of virion sense gene expression in wheat dwarf virus.

We have used particle bombardment (biolistics) to deliver replication-competent wheat dwarf virus (WDV)-based constructs, carrying reporter gene sequences fused to the virion sense promoter (Pv) or the CaMV 35S promoter, to suspension culture cells and immature zygotic embryos of wheat. While the replication of WDV double-stranded DNA forms (replicons) was equivalent between wheat suspension culture cells and embryos, GUS reporter gene activity was 20-40 times higher in the embryo cultures. Maximum expression of WDV replicons occurred in the embryonic axis tissue of wheat embryos but their expression in suspension cells was compromised, compared with transiently maintained input plasmid DNA containing the same sequences. From these studies, we propose that WDV replicons are subject to a host cell-controlled competency for virion sense transcription. The term competency is used to distinguish between the phenomenon described here and control of gene expression by specific transcription factors. Control of competency is independent of Pv, the replacement 35S promoter and of the complementary sense control of virion sense expression involving specific sequences in Pv. We propose that factors controlling the competency for replicon expression may be present in cells which, as well as maintaining high rates of DNA synthesis, are totipotent. Cell type control of active chromatin, methylation of specific sequences in WDV minichromosomes and/or interaction of virus-encoded proteins with specific host factors are considered as possible mechanisms.

Identification of cDNAS encoding plastid-targeted glutathione peroxidase.

A cDNA was isolated from pea leaf RNA which encodes a phospholipid hydroperoxide glutathione peroxidase (PHGPX; E.C. 1.1.1.1.9). The N-terminal section of this PHGPX encodes a recognisable chloroplast transit peptide. Efficient import in vitro of the pre-PHGPX protein into the stroma of isolated pea chloroplasts confirmed that the PHGPX is a chloroplast-located enzyme. The pea PHGPX has highly conserved homologues in Arabidopsis, citrus and Nicotiana sylvestris and the authors suggest that these proteins are also localised in the chloroplast and not in the cytosol as previously supposed.

Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress.

Exposure of Arabidopsis plants that were maintained under low light (200 mumol of photons m-2 sec-1) to excess light (2000 mumol of photons m-2 sec-1) for 1 hr caused reversible photoinhibition of photosynthesis. Measurements of photosynthetic parameters and the use of electron transport inhibitors indicated that a novel signal transduction pathway was initiated at plastoquinone and regulated, at least in part, by the redox status of the plastoquinone pool. This signal, which preceded the photooxidative burst of hydrogen peroxide (H2O2) associated with photoinhibition of photosynthesis, resulted in a rapid increase (within 15 min) in mRNA levels of two cytosolic ascorbate peroxidase genes (APX1 and APX2). Treatment of leaves with exogenous reduced glutathione abolished this signal, suggesting that glutathione or the redox status of the glutathione pool has a regulatory impact on this signaling pathway. During recovery from photooxidative stress, transcripts for cytosolic glutathione reductase (GOR2) increased, emphasizing the role of glutathione in this stress.

Cloning and characterisation of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress.

A second glutathione reductase (GR) cDNA has been cloned and sequenced from pea (Pisum sativum L. cv. Birte). This new GR cDNA (GOR2) does not encode a pre-protein with a transit peptide and therefore is most likely to represent a cytosolic GR. It is significantly different at the DNA level from the previously cloned chloroplastidial/mitochondrial pea GR (GOR1), but retains the features characteristic of GRs from all sources and has GR activity when expressed in Escherichia coli. GOR2 maps to linkage group 6 on the pea genome map and it seems likely that this is the only locus for this gene. In contrast to GOR1, transcript levels of GOR2 increase in the recovery (post-stress) phases of both drought and chilling by about ten- and three-fold respectively. GOR2 therefore may play a role in the restoration of the post-stress redox state of the cytosolic glutathione pool.

The two nonstructural proteins from wheat dwarf virus involved in viral gene expression and replication are retinoblastoma-binding proteins.

Tumor-inducing viruses like simian virus 40 or the human adenovirus produce oncoproteins which interfere with the cellular retinoblastoma (Rb) tumor-suppressor protein to create an appropriate molecular environment in the nucleus for viral transcription and replication. Such a strategy has been considered to be restricted to animal viruses. Here we demonstrate that plant viruses may use similar mechanisms for recruiting host factors. Wheat dwarf virus (WDV) encodes two potential nonstructural proteins, C1 and C1:C2, both containing the consensus Rb-binding motif LeuXCysXGlu that allows the oncoproteins from animal viruses to inactivate Rb. C1:C2 is a key determinant of viral replication and V(virion)-sense expression. Using a yeast two-hybrid protein assay, we demonstrate for the first time that the C1:C2 protein from WDV interacts with a retinoblastoma protein, providing an explanation for the previously observed dependence of viral replication on an intact Rb-binding motif. We also show that C1, for which no function had been demonstrated, is required for V-sense gene expression. This suggests that V-sense expression might be dependent on the interaction of C1 with Rb. Our findings provide further evidence for the presence of transforming-like proteins in a plant virus and will help to explain the production of symptoms in a plant viral infection through a mechanism mediated by a key regulator of cell cycle and differentiation.

Cloning and characterisation of glutathione reductase cDNAs and identification of two genes encoding the tobacco enzyme.

We have isolated 4 cDNA clones (GRT1-4) encoding glutathione reductase (GR) from a tobacco (Nicotiana tabacum L.) leaf cDNA library. The cDNAs were almost identical: GRT1, GRT3 and GRT4 represented the same gene, differing only in that GRT4 contained an intron within the C-terminal part of the coding sequence. Failure to splice out this intron resulted in a substitution of the final 13 amino acids of the deduced amino acid sequence. A second gene was represented by GRT2. Southern blots indicated that there were two related GR genes in tobacco. The presence of multiple isoforms of GR in tobacco may be explained in part by the expression of a small gene family. In addition, alternative isoforms may result from translation of different mRNAs derived from the same gene by intron skipping during the splicing of nascent GR mRNAs.

Digitaria streak geminivirus replicative forms are abundant in S-phase nuclei of infected cells.

We have analyzed the replication of Digitaria streak geminivirus (DSV) using flow cytometry. Nuclei from infected plants were sorted on the basis of their DNA content in the different phases of the cell cycle and viral DNA forms in them were analyzed. DSV replicative forms were much more abundant (up to 20 times in one experiment) in S-phase nuclei than in G0-G1 and G2 nuclei, while single-stranded viral DNA did not show such dramatic increase during any phase. DSV replicative forms constitute almost 90% of total viral DNA forms in S-phase nuclei. DSV replication seems to be synchronized with host DNA replication and might be primarily under control of host factors becoming available only at the G1/S boundary.

Mutagenesis of the virion-sense open reading frames of tomato leaf curl geminivirus.

A series of frame shift, deletion, and inversion mutants in the virion-sense open reading frames (ORFs) of the monopartite geminivirus tomato leaf curl virus have been constructed and their ability to replicate, produce single-stranded DNA, spread, and cause symptoms in tomato plants has been investigated. Disruptions in the V1 ORF lead to symptomless, systemic infections with a reduced titer of all viral DNA forms while interruptions in the V2 (coat protein) ORF disrupted spread of the virus. Mutagenesis of the virion-sense ORFs did not affect the replication of viral double-stranded DNA, although both V1 and V2 products appear to play a role in the accumulation of viral single-stranded DNA.

Mapping of the polycistronic RNAs of tomato leaf curl geminivirus.

Four major transcripts from infected tomato were identified and mapped onto the monopartite genome of the geminivirus tomato leaf curl virus (TLCV). The C1, C2, and C3 ORFs are spanned by one transcript and a second internal RNA covered C2 and C3. Both these RNAs have their counterparts in the DNA A components of the bipartite subgroup of geminiviruses. The 5' ends of the virion-sense RNAs map either side of the first in-frame AUG of the V1 ORF. The 3' ends of the virion-sense RNAs are coterminal and overlap with the 3' ends of the complementary-sense RNAs. All of the RNAs have transcription regulatory sequences close to their mapped termini and the presence of overlapping transcripts suggests that temporal regulation of their synthesis may occur. The translation of these polycistronic RNAs is discussed in the light of the RNA mapping data.

Nucleotide sequence and genome organization of tomato leaf curl geminivirus.

The genome of tomato leaf curl virus (TLCV) from Australia was cloned and its complete nucleotide sequence determined. It is a single circular ssDNA of 2766 nucleotides containing the consensus nonanucleotide sequence present in all geminiviruses. It has six open reading frames with an organization resembling that of certain other dicotyledonous plant-infecting monopartite geminiviruses, i.e. tomato yellow leaf curl and beet curly top viruses. The regulatory sequences present indicate a bidirectional mode of transcription. A dimeric TLCV DNA clone was constructed in a binary vector and used to agroinoculate three different host species. Typical virus infections were produced, confirming that the single DNA component is sufficient for infectivity.

Coordinate regulation of replication and virion sense gene expression in wheat dwarf virus.

We have investigated the relationship between viral DNA replication and virion sense gene expression in wheat dwarf virus (WDV), a member of the geminivirus group, by testing a series of deletion mutants in transfected Triticum monococcum (einkorn) protoplasts. Mutants contained a transcription fusion of the chloramphenicol acetyltransferase coding sequence to the virion sense promoter that replaced the viral coat protein coding sequence. The deletion analysis revealed that WDV replication and virion sense transcription can proceed independently and are controlled in part by nonoverlapping elements in the large intergenic region. These data and those from a C2 open reading frame (ORF) frameshift mutant also showed that the product of the C2 ORF (C1-C2 protein) is independently involved in both DNA replication and activation of the virion sense promoter. The amino acid sequences encoded by C2, which are highly conserved in the geminivirus group, show some homology to the DNA binding domain of the myb-related class of plant transcription factors. The possible involvement of the host in controlling the function of the C1-C2 protein and the implication of these data for the development of WDV-based gene vectors are discussed.