Poplar is one of the most important tree species in the north temperate zone, but poplar plantations are quite water intensive. We report here that CaMV 35S promoter-driven overexpression of the PdERECTA gene, which is a member of the LRR-RLKs family from Populus nigra × (Populus deltoides × Populus nigra), improves water use efficiency and enhances drought tolerance in triploid white poplar. PdERECTA localizes to the plasma membrane. Overexpression plants showed lower stomatal density and larger stomatal size. The abaxial stomatal density was 24–34% lower and the stomatal size was 12–14% larger in overexpression lines. Reduced stomatal density led to a sharp restriction of transpiration, which was about 18–35% lower than the control line, and instantaneous water use efficiency was around 14–63% higher in overexpression lines under different conditions. These phenotypic changes led to increased drought tolerance. PdERECTA overexpression plants not only survived longer after stopping watering but also performed better when supplied with limited water, as they had better physical and photosynthesis conditions, faster growth rate, and higher biomass accumulation. Taken together, our data suggest that PdERECTA can alter the development pattern of stomata to reduce stomatal density, which then restricts water consumption, conferring enhanced drought tolerance to poplar. This makes PdERECTA trees promising candidates for establishing more water use efficient plantations.

Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔC_t, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.

Heat shock transcription factors (HSFs) are crucial components in heat stress response. However, the contribution of the HSFs governing the inherent thermotolerance in Rhodomyrtus tomentosa has barely been investigated. We here compared the roles of RtHSFA9a, RtHSFA9b, and RtHSFA9c in heat stress tolerance. These three genes are the results of gene duplication events, but there exist vast variations in their amino acid sequences. They are all localized to the nucleus. Arabidopsis thaliana plants with overexpressed RtHSFA9a and RtHSFA9c outperformed the wild-type plants, while the over-accumulation of RtHSFA9b had little impact on plant thermotolerance. By transiently overexpressing RtHSFA9a, RtHSFA9b, and RtHSFA9c in R. tomentosa seedlings, the mRNA abundance of heat shock response genes, including RtHSFA2a, RtHSFA2b, RtHSP17.4, RtHSP21.8, RtHSP26.5, and RtHSP70, were upregulated. Transactivation assays confirmed that there exist regulatory divergences among these three genes, viz., RtHSFA9a has the highest transcription activity in regulating RtHSFA2a, RtHSFA2b, RtHSP21.8, and RtHSP70; RtHSFA9c can transcriptionally activate RtHSFA2b, RtHSP21.8, and RtHSP70; RtHSFA9b makes limited contributions to the accumulation of RtHSFA2b, RtHSP21.8, and RtHSP70. Our results indicate that the RtHSFA9 genes make crucial contributions to the thermal adaption of R. tomentosa by positively regulating the RtHSFA2a, RtHSFA2b, and RtHSP genes, which provides novel insights into the RtHSFA9 subfamily.

Ionic aluminum (Al) forms in acidic soils and inhibits plant growth, even at low concentrations. Rose myrtle (Rhodomyrtus tomentosa), a shrub native to tropical and subtropical regions, thrives in acidic-Al soils. Here, we found that mild concentrations of Al promote rose myrtle growth. Transcriptomic disturbances induced by low or high Al stress were predominantly nonoverlapping in the species. Mild Al stress (0.1 mM Al3+) enhanced rose myrtle root elongation through the upregulation of xyloglucan metabolism, nutrient uptake and utilization, and auxin transport. In contrast, high Al stress (1 mM Al3+) activated detoxification pathways, including the secretion of organic acid and glutathione metabolism. Members of the aluminum-activated malate transporter (ALMT) family, particularly the conserved RtALMT11 and variable RtALMT18, play a pivotal role in Al tolerance. Heterologous expression of RtALMT11 and RtALMT18 complemented the Al-sensitive phenotype of almt1-KO Arabidopsis (Arabidopsis thaliana). High Al3+ induced the expression of RtALMT11, mediating the synthesis of callose, which may serve as a physical barrier to mitigate Al penetration and facilitate vacuolar Al sequestration. RtALMT18 pre-emptively regulated internal defense in the stele independently of aluminum load, while also functioning as a proton/malate transporter. Beyond enhancing Al tolerance, RtALMT18 promoted the growth of transgenic Arabidopsis and poplar (Populus alba×Populus glandulosa, “84K”). The functional divergence within the ALMT family reveals distinct roles in promoting the growth of rose myrtle under low Al conditions and during the high-Al detoxification process. These findings uncover Al's dual role as both a growth promoter and stress inducer, offering insights for developing Al-tolerant crops and rehabilitating acidic soils.