Histological and single-nucleus transcriptome analyses reveal the specialized functions of ligular sclerenchyma cells and key regulators of leaf angle in maize.

Leaf angle (LA) is a crucial factor that affects planting density and yield in maize. However, the regulatory mechanisms underlying LA formation remain largely unknown. In this study, we performed a comparative histological analysis of the ligular region across various maize inbred lines and revealed that LA is significantly influenced by a two-step regulatory process involving initial cell elongation followed by subsequent lignification in the ligular adaxial sclerenchyma cells (SCs). Subsequently, we performed both bulk and single-nucleus RNA sequencing, generated a comprehensive transcriptomic atlas of the ligular region, and identified numerous genes enriched in the hypodermal cells that may influence their specialization into SCs. Furthermore, we functionally characterized two genes encoding atypical basic-helix-loop-helix (bHLH) transcription factors, bHLH30 and its homolog bHLH155, which are highly expressed in the elongated adaxial cells. Genetic analyses revealed that bHLH30 and bHLH155 positively regulate LA expansion, and molecular experiments demonstrated their ability to activate the transcription of genes involved in cell elongation and lignification of SCs. These findings highlight the specialized functions of ligular adaxial SCs in LA regulation by restricting further extension of ligular cells and enhancing mechanical strength. The transcriptomic atlas of the ligular region at single-nucleus resolution not only deepens our understanding of LA regulation but also enables identification of numerous potential targets for optimizing plant architecture in modern maize breeding.

Long noncoding RNA from Betula platyphylla, BplncSIR1, confers salt tolerance by regulating BpNAC2 to mediate reactive oxygen species scavenging and stomatal movement.

Long noncoding RNAs (lncRNAs) play an important role in abiotic stress tolerance. However, their function in conferring abiotic stress tolerance is still unclear. Herein, we characterized the function of a salt-responsive nuclear lncRNA (BplncSIR1) from Betula platyphylla (birch). Birch plants overexpressing and knocking out for BplncSIR1 were generated. BplncSIR1 was found to improve salt tolerance by inducing antioxidant activity and stomatal closure, and also accelerate plant growth. Chromatin isolation by RNA purification (ChIRP) combined with RNA sequencing indicated that BplncSIR1 binds to the promoter of BpNAC2 (encoding NAC domain-containing protein 2) to activate its expression. Plants overexpressing and knocking out for BpNAC2 were generated. Consistent with that of BplncSIR1, overexpression of BpNAC2 also accelerated plant growth and conferred salt tolerance. In addition, BpNAC2 binds to different cis-acting elements, such as G-box and 'CCAAT' sequences, to regulate the genes involved in salt tolerance, resulting in reduced ROS accumulation and decreased water loss rate by stomatal closure. Taken together, BplncSIR1 serves as the regulator of BpNAC2 to induce its expression in response to salt stress, and activated BpNAC2 accelerates plant growth and improves salt tolerance. Therefore, BplncSIR1 might be a candidate gene for molecular breeding to cultivate plants with both a high growth rate and improved salt tolerance.

Identification of birch lncRNAs and mRNAs responding to salt stress and characterization of functions of lncRNA.

Long noncoding RNAs (lncRNAs) are important in abiotic stress tolerance. Here, we identified salt-responsive genes and lncRNAs in the roots and leaves of Betula platyphylla Suk. (birch), and characterized their lncRNAs functions. In total, 2660 mRNAs and 539 lncRNAs responding to salt treatment were identified using RNA-seq. The salt-responsive genes were substantially enriched in 'cell wall biogenesis' and 'wood development' in the roots and were enriched in 'photosynthesis' and 'response to stimulus' in the leaves. Meanwhile, the potential target genes of the salt-responsive lncRNAs in roots and leaves were both enriched in 'nitrogen compound metabolic process' and 'response to stimulus'. We further built a method for quickly identifying abiotic stress tolerance of lncRNAs, which employed transient transformation for overexpression and knock-down of the lncRNA, enabling gain- and loss-of-function analysis. Using this method, 11 randomly selected salt-responsive lncRNAs were characterized. Among them, six lncRNAs confer salt tolerance, two lncRNAs confer salt sensitivity, and the other three lncRNAs are not involved in salt tolerance. In addition, a lncRNA, LncY1, was further characterized, which improves salt tolerance by regulating two transcription factors, BpMYB96 and BpCDF3. Taken together, our results suggested that lncRNAs play important roles in the salt response of birch plants.

Hierarchical transcription factor and regulatory network for drought response in Betula platyphylla.

Although many genes and biological processes involved in abiotic stress response have been identified, how they are regulated remains largely unclear. Here, to study the regulatory mechanism of birch (Betula platyphylla) responding to drought induced by polyethylene glycol (PEG) 6000 (20%, w/v), a partial correlation coefficient-based algorithm for constructing gene regulatory network (GRN) was proposed, and a three-layer hierarchical GRN was constructed, including 68 transcription factors (TFs), and 252 structural genes. Totally, 1448 predicted regulatory relationships are included, and most of them are novel. The reliability of GRN was verified by ChIP-PCR and qRT-PCR based on transient transformation. About 55% of genes in the bottom layer of GRN could confer drought tolerance. We selected the two TFs, BpMADS11 and BpNAC090, from the up layer and characterized their function in drought tolerance. Overexpression of BpMADS11 and BpNAC090 both reduces electrolyte leakage, ROS and MDA contents, displaying increased drought tolerance than wild-type birch. According to this GRN, the important biological processes involved in drought were identified, including "signaling hormone pathways", "water transport", "regulation of stomatal movement" and "response to oxidative stress". This work indicated that BpERF017, BpAGL61 and BpNAC090 are the key upstream regulators in birch drought tolerance. Our data clearly revealed the upstream regulators and TF-DNA interaction regulate different biological processes to adapt drought stress.

Characterization of the Function of Two S1Fa-Like Family Genes From Populus trichocarpa.

S1Fa-like transcription factors (TFs) are small molecular weight proteins that contain both nuclear localization and DNA binding domains. However, the functions of S1Fa-like TFs are poorly understood. In the present study, we identified the S1Fa-like TFs from the Populus trichocarpa genome, which revealed two S1Fa-like TF genes, PtS1Fa1 and PtS1Fa2. PtS1Fa1 and PtS1Fa2 expression was suppressed by drought and salt stress, and was also significantly altered by ABA, MeJA, or SA treatment. Both PtS1Fa1 and PtS1Fa2 are nuclear proteins. Transgenic P. trichocarpa plants overexpressing PtS1Fa1 and PtS1Fa2, respectively, were generated. The plants overexpressing PtS1Fa2 showed increased fresh weight, chlorophyll content, and root length and weight compared with those in wild-type (WT) P. trichocarpa under drought conditions. Meanwhile, these phenotype traits of plants overexpressing PtS1Fa1 were similar to those of WT plants. Furthermore, overexpression of PtS1Fa2 reduced the malondialdehyde (MDA) content, electrolyte leakage, H2O2 and O2- contents, and increased superoxide dismutase (SOD) and peroxidase (POD) activities. The expression of SOD and POD was also induced by PtS1Fa2. However, overexpression of PtS1Fa1 failed to affect any of these physiological parameters or SOD and POD gene expression. These results suggested that PtS1Fa2 plays a role in drought tolerance, and confers drought tolerance by increase antioxidant activity to reduce reactive oxygen species (ROS) accumulation.