Activation tagging identifies Arabidopsis transcription factor AtMYB68 for heat and drought tolerance at yield determining reproductive stages.

Heat stress occurring at reproductive stages can result in significant and permanent damage to crop yields. However, previous genetic studies in understanding heat stress response and signaling were performed mostly on seedling and plants at early vegetative stages. Here we identify, using a developmentally defined, gain-of-function genetic screen with approximately 18 000 Arabidopsis thaliana activation-tagged lines, a mutant that maintained productive seed set post-severe heat stress during flowering. Genome walking indicated this phenotype was caused by the insertion of 35S enhancers adjacent to a nuclear localized transcription factor AtMYB68. Subsequent overexpression analysis confirmed that AtMYB68 was responsible for the reproductive heat tolerance of the mutant. Furthermore, these transgenic Arabidopsis plants exhibited enhanced abscisic acid sensitivity at and post-germination, reduced transpirational water loss during a drought treatment, and enhanced seed yield under combined heat and drought stress during flowering. Ectopic expression of AtMYB68 in Brassica napus driven either by 35S or by heat-inducible promoter recapitulated the enhanced reproductive heat stress and drought tolerance phenotypes observed in the transgenic Arabidopsis. The improvement to heat stress is likely due to enhanced pollen viability observed in the transgenic plants. More importantly, the transgenic canola showed significant yield advantages over the non-transgenic controls in multiple locations, multiple season field trials under various drought and heat stress conditions. Together these results suggest that AtMYB68 regulate plant stress tolerance at the most important yield determining stage of plant development, and is an effective target for crop yield protection under current global climate volatility.

Narrowing down the targets: towards successful genetic engineering of drought-tolerant crops.

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.

Shoot-specific down-regulation of protein farnesyltransferase (alpha-subunit) for yield protection against drought in canola.

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.

Molecular tailoring of farnesylation for plant drought tolerance and yield protection.

Protecting 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 beta-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 alpha- or beta-subunit 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 non-transgenic 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 deficit-induced 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.

A pea antisense gene for the chloroplast stromal processing peptidase yields seedling lethals in Arabidopsis: survivors show defective GFP import in vivo.

The stromal processing peptidase (SPP) of chloroplasts is a metalloendopeptidase that cleaves in vitro a broad range of precursor substrates. Here, we have investigated SPP's role in vivo. Two pea cDNA antisense constructs encoding either full-length SPP (AS4.0) or its N-terminal half (AS2.2) are introduced into Arabidopsis, which contains one gene for SPP that codes for one isoform. Our analyses show that AS4.0 produces a strong mutant phenotype, with a large percentage of the plants dying as seedling lethals. Surviving plants exhibited slower shoot and root growth, and grossly aberrant leaf morphology. Green and white sectoring, and purple pigmentation was observed. In cells where chloroplasts could be identified, they were fewer in number by at least 40%, thylakoids were not fully developed, and starch granules accumulated. The phenotype produced by AS2.2 was less severe. Using green fluorescent protein (GFP) fused to a transit peptide as a reporter, we examined import into chloroplasts in vivo. In the Arabidopsis antisense lines, GFP was located primarily in the cytosol, indicating that an early step in the import pathway was impeded. In a tobacco AS14 line expressing AS2.2, GFP was located in the cytosol, on the envelope, and in the stroma. The three patterns were observed in different cells, suggesting that the import capacity of individual cells was not the same. Our in vivo studies demonstrate that SPP is essential for chloroplast biogenesis and plant survival. SPP does not act independently in the stroma, but its activity influences earlier steps in the import pathway.