Drought is the most important environmental stress affecting agriculture worldwide. Exploiting yield potential and maintaining yield stability of crops in water-limited environments are urgent tasks that must be undertaken in order to guarantee food supply for the increasing world population. Tremendous efforts have been devoted to identifying key regulators in plant drought response through genetic, molecular, and biochemical studies using, in most cases, the model species Arabidopsis thaliana. However, only a small portion of these regulators have been explored as potential candidate genes for their application in the improvement of drought tolerance in crops. Based on biological functions, these genes can be classified into the following three categories: (1) stress-responsive transcriptional regulation (e.g. DREB1, AREB, NF-YB); (2) post-transcriptional RNA or protein modifications such as phosphorylation/dephosphorylation (e.g. SnRK2, ABI1) and farnesylation (e.g. ERA1); and (3) osomoprotectant metabolism or molecular chaperones (e.g. CspB). While continuing down the path to discovery of new target genes, serious efforts are also focused on fine-tuning the expression of the known candidate genes for stress tolerance in specific temporal and spatial patterns to avoid negative effects in plant growth and development. These efforts are starting to bear fruit by showing yield improvements in several crops under a variety of water-deprivation conditions. As most such evaluations have been performed under controlled growth environments, a gap still remains between early success in the laboratory and the application of these techniques to the elite cultivars of staple crops in the field. Nevertheless, significant progress has been made in the identification of signaling pathways and master regulators for drought tolerance. The knowledge acquired will facilitate the genetic engineering of single or multiple targets and quantitative trait loci in key crops to create commercial-grade cultivars with high-yielding potential under both optimal and suboptimal conditions.

). † These authors contributed equally to this work. SummaryProtecting crop yield under drought stress is a major challenge for modern agriculture. One biotechnological target for improving plant drought tolerance is the genetic manipulation of the stress response to the hormone abscisic acid (ABA). Previous genetic studies have implicated the involvement of the b-subunit of Arabidopsis farnesyltransferase (ERA1) in the regulation of ABA sensing and drought tolerance. Here we show that molecular manipulation of protein farnesylation in Arabidopsis, through downregulation of either the a-or bsubunit of farnesyltransferase enhances the plant's response to ABA and drought tolerance. To test the effectiveness of tailoring farnesylation in a crop plant, transgenic Brassica napus carrying an ERA1 antisense construct driven by a drought-inducible rd29A promoter was examined. In comparison with the nontransgenic control, transgenic canola showed enhanced ABA sensitivity, as well as significant reduction in stomatal conductance and water transpiration under drought stress conditions. The antisense downregulation of canola farnesyltransferase for drought tolerance is a conditional and reversible process, which depends on the amount of available water in the soil. Furthermore, transgenic plants were more resistant to water deficitinduced seed abortion during flowering. Results from three consecutive years of field trial studies suggest that with adequate water, transgenic canola plants produced the same amount of seed as the parental control. However, under moderate drought stress conditions at flowering, the seed yields of transgenic canola were significantly higher than the control. Using protein farnesyltransferase as an effective target, these results represent a successful demonstration of engineered drought tolerance and yield protection in a crop plant under laboratory and field conditions.

Canola (Brassica napus L.) is one of the most important oilseed crops in the world and its seed yield and quality are significantly affected by drought stress. As an innate and adaptive response to water deficit, land plants avoid potential damage by rapid biosynthesis of the phytohormone abscisic acid (ABA), which triggers stomatal closure to reduce transpirational water loss. The ABA-mediated stomatal response is a dosage-dependent process; thus, one genetic engineering approach for achieving drought avoidance could be to sensitize the guard cell's responsiveness to this hormone. Recent genetic studies have pinpointed protein farnesyltransferase as a key negative regulator controlling ABA sensitivity in the guard cells. We have previously shown that down-regulation of the gene encoding Arabidopsis beta-subunit of farnesyltransferase (ERA1) enhances the plant's sensitivity to ABA and drought tolerance. Although the alpha-subunit of farnesyltransferase (AtFTA) is also implicated in ABA sensing, the effectiveness of using such a gene target for improving drought tolerance in a crop plant has not been validated. Here, we report the identification and characterization of the promoter of Arabidopsis hydroxypyruvate reductase (AtHPR1), which expresses specifically in the shoot and not in non-photosynthetic tissues such as root. The promoter region of AtHPR1 contains the core motif of the well characterized dehydration-responsive cis-acting element and we have confirmed that AtHPR1 expression is inducible by drought stress. Conditional and specific down-regulation of FTA in canola using the AtHPR1 promoter driving an RNAi construct resulted in yield protection against drought stress in the field. Using this molecular strategy, we have made significant progress in engineering drought tolerance in this important crop species.

The in vitro import characteristics of six different precursors of plastid proteins were assessed to determine differences in the protein import pathways of leucoplasts and chloroplasts. Five of these precursor proteins are destined to different subchloroplast sites, and one is a leucoplast stromal precursor protein. The results indicate that some of these precursors can be imported equally into both plastid types and others preferentially into one type of plastid versus the other. The ability of plastids to import different proteins correlates with the in vivo steady state levels of these proteins. Additional differences were also observed in the intraorganellar portion of the translocation pathway for two thylakoidal proteins. The differences in import characteristics were found to be predominantly governed by information in the transit peptides, since attachment of the various transit peptides to different plastid and foreign proteins demonstrated that the import behavior of the proteins is transferable with the transit sequence. These results indicate that the import mechanisms of leucoplasts and chloroplasts are sufficiently different such that the plastids respond differently to the information present in the transit peptides.The NH 2 -terminal transit peptide extension of nuclear-encoded precursors of plastid proteins is considered the primary signal for directing posttranslational import of the precursor (see reviews by Keegstra, 1989;Keegstra et al., 1989). The transit peptide generally possesses sufficient information for the correct targeting of proteins to the plastid compartment and for subsequent intraorganellar sorting. Targeting of stromal-destined precursor proteins appears to be directed by information in a contiguous segment of NH 2 -terminal residues (Smeekens et al., 1986Schmidt and Mishkind, 1986;Van den Broeck et al., 1985), whereas thylakoid lumen proteins, e.g. plastocyanin and Oee1, 1 are directed by bipartite transit peptides (Smeekens et al., 1986Weisbeek et al., 1987;de Boer et al., 1988;Ko and Cashmore, 1989). There are also examples of proteins such as Cab and Ferrodoxin binding subunit of photosystem I that possess suborganellar targeting information within the mature region of the protein rather than within the transit peptide (Cline, 1988;Lamppa, 1988;Van den Broeck et al., 1988;Viitanen et al., 1988;Hand et al., 1989; also see review by Theg and Scott, 1993). In these cases, the NH 2 -terminal transit peptide acts only to import the protein into the stromal compartment.The targeting of proteins into plastids other than chloroplasts and the additional information accompanying this process are even more complex when it is considered that plastids can vary enormously in function. Most of the evidence to date indicates that targeting information for other types of plastids can be transposed from chloroplastic precursor proteins without apparent effect. For instance, the chloroplast Rbcs precursor proteins can be imported into leucoplasts (Boyle et al., 1986; Halpin et al., 1989), ...