A goal of modern agriculture is to improve plant drought tolerance and production per amount of water used, referred to as water use efficiency (WUE). Although stomatal density has been linked to WUE, the causal molecular mechanisms have yet to be determined. Arabidopsis thaliana GT-2 LIKE 1 (GTL1) loss-of-function mutations result in increased water deficit tolerance and higher integrated WUE by reducing daytime transpiration without a demonstrable reduction in biomass accumulation. gtl1 plants had higher instantaneous WUE that was attributable to ;25% lower transpiration and stomatal conductance but equivalent CO 2 assimilation. Lower transpiration was associated with higher STOMATAL DENSITY AND DISTRIBUTION1 (SDD1) expression and an ;25% reduction in abaxial stomatal density. GTL1 expression occurred in abaxial epidermal cells where the protein was localized to the nucleus, and its expression was downregulated by water stress. Chromatin immunoprecipitation analysis indicated that GTL1 interacts with a region of the SDD1 promoter that contains a GT3 box. An electrophoretic mobility shift assay was used to determine that the GT3 box is necessary for the interaction between GTL1 and the SDD1 promoter. These results establish that GTL1 negatively regulates WUE by modulating stomatal density via transrepression of SDD1.

Diminishing global fresh water availability has focused research to elucidate mechanisms of water use in poplar, an economically important species. A GT-2 family trihelix transcription factor that is a determinant of water use efficiency (WUE), PtaGTL1 (GT-2 like 1), was identified in Populus tremula × P. alba (clone 717-IB4). Like other GT-2 family members, PtaGTL1 contains both N- and C-terminal trihelix DNA binding domains. PtaGTL1 expression, driven by the Arabidopsis thaliana AtGTL1 promoter, suppressed the higher WUE and drought tolerance phenotypes of an Arabidopsis GTL1 loss-of-function mutation (gtl1-4). Genetic suppression of gtl1-4 was associated with increased stomatal density due to repression of Arabidopsis STOMATAL DENSITY AND DISTRIBUTION1 (AtSDD1), a negative regulator of stomatal development. Electrophoretic mobility shift assays (EMSA) indicated that a PtaGTL1 C-terminal DNA trihelix binding fragment (PtaGTL1-C) interacted with an AtSDD1 promoter fragment containing the GT3 box (GGTAAA), and this GT3 box was necessary for binding. PtaGTL1-C also interacted with a PtaSDD1 promoter fragment via the GT2 box (GGTAAT). PtaSDD1 encodes a protein with 60% primary sequence identity with AtSDD1. In vitro molecular interaction assays were used to determine that Ca2+-loaded calmodulin (CaM) binds to PtaGTL1-C, which was predicted to have a CaM-interaction domain in the first helix of the C-terminal trihelix DNA binding domain. These results indicate that, in Arabidopsis and poplar, GTL1 and SDD1 are fundamental components of stomatal lineage. In addition, PtaGTL1 is a Ca2+-CaM binding protein, which infers a mechanism by which environmental stimuli can induce Ca2+ signatures that would modulate stomatal development and regulate plant water use.

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Eutrema salsugineum is a model species for the study of plant adaptation to abiotic stresses. Two accessions of E. salsugineum, Shandong (SH) and Yukon (YK), exhibit contrasting morphology, biotic, and abiotic stress tolerance. Transcriptome and metabolic profiling from tissue samples collected during the dark period were used to investigate the molecular and metabolic bases of these contrasting phenotypes. RNA sequencing identified 17,888 expressed genes, of which 157 were not in the published reference genome and 65 were detected for the first time. Differential expression was detected for only 31 genes. The RNA sequencing data contained 14,808 single nucleotide polymorphisms (SNPs) in transcripts, 3,925 of which are newly identified. Among the differentially expressed genes, there were no obvious candidates for the physiological or morphological differences between SH and YK. Metabolic profiling indicated that YK accumulates free fatty acids and long-chain fatty acid derivatives as compared to SH; whereas sugars are more abundant in SH. Metabolite levels suggest that carbohydrate and respiratory metabolism, including starch degradation, is more active during the first half of the dark period in SH. These metabolic differences may explain the greater biomass accumulation in YK over SH. The accumulation of 56% of the identified metabolites was lower in F1 hybrids than the mid-parent averages and the accumulation of 17% of the metabolites in F1 plants transgressed the level in both parents. Concentrations of several metabolites in F1 hybrids agree with previous studies and suggest a role for primary metabolism in heterosis. The improved annotation of the E. salsugineum genome and newly-identified high-quality SNPs will permit accelerated studies using the standing variation in this species to elucidate the mechanisms of its diverse adaptations to the environment.

The North American native plants Spiraea alba and S. tomentosa have potential as landscape plants because they are small- to medium-sized shrubs with showy flowers that persist from early to late summer. However, the cultural requirements of these species are not well documented. Both species grow in slightly acidic soils in their native habitats. The objective of this work was to determine if the growth and/or appearance of these shrubs is affected by the neutral to slightly alkaline soils common throughout the Midwest. Spiraea alba grown from three seed sources and S. tomentosa grown from a single seed source were grown for 2 years in soils of different pH. Elemental sulfur (S) was incorporated into a Drummer silty clay loam with a pH of 7.2 to bring the pH to 5.8 and 6.4 2 years after incorporation, thus establishing plots with three pH levels for assessing growth and appearance. The soil pH increased to 6.4 and 6.8 in the amended plots between Years 2 and 3 after S incorporation. Soil concentrations of exchangeable magnesium (Mg) and calcium (Ca) were lowered with added S. Height and width varied among S. alba from different seed sources, and height and width of all plants were reduced when grown at the highest soil pH. Leaf greenness, specific leaf weight, and individual leaf area were unaffected by pH. Leaf concentrations of nitrogen, potassium, zinc, and manganese were higher in lower soil pH, whereas Mg was lowest at the lowest soil pH. These results suggest that S. alba and S. tomentosa can be grown in neutral pH soils without an effect on plant appearance, but plant size will be less than at pH levels closer to the native soils for these species. Although some micronutrients were present at lower concentrations at the neutral pH, leaf greenness was unaffected, again suggesting that these plants may perform suitably outside of their native habitat pH range.