Genome-Wide Identification, Characterization, and Expression Analysis of the HD-Zip Gene Family in Lagerstroemia for Regulating Plant Height.

The Homeodomain leucine zipper (HD-Zip) family of transcription factors is crucial in helping plants adapt to environmental changes and promoting their growth and development. Despite research on the HD-Zip family in various plants, studies in Lagerstroemia (crape myrtle) have not been reported. This study aimed to address this gap by comprehensively analyzing the HD-Zip gene family in crape myrtle. This study identified 52 HD-Zip genes in the genome of Lagerstroemia indica, designated as LinHDZ1-LinHDZ52. These genes were distributed across 22 chromosomes and grouped into 4 clusters (HD-Zip I-IV) based on their phylogenetic relationships. Most gene structures and motifs within each cluster were conserved. Analysis of protein properties, gene structure, conserved motifs, and cis-acting regulatory elements revealed diverse roles of LinHDZs in various biological contexts. Examining the expression patterns of these 52 genes in 6 tissues (shoot apical meristem, tender shoot, and mature shoot) of non-dwarf and dwarf crape myrtles revealed that 2 LinHDZs (LinHDZ24 and LinHDZ14) and 2 LinHDZs (LinHDZ9 and LinHDZ35) were respectively upregulated in tender shoot of non-dwarf crape myrtles and tender and mature shoots of dwarf crape myrtles, which suggested the important roles of these genes in regulate the shoot development of Lagerstroemia. In addition, the expression levels of 2 LinHDZs (LinHDZ23 and LinHDZ34) were significantly upregulated in the shoot apical meristem of non-dwarf crape myrtle. These genes were identified as key candidates for regulating Lagerstroemia plant height. This study enhanced the understanding of the functions of HD-Zip family members in the growth and development processes of woody plants and provided a theoretical basis for further studies on the molecular mechanisms underlying Lagerstroemia plant height.

Genome-wide analysis of the heat shock transcription factor family reveals saline-alkali stress responses in Xanthoceras sorbifolium.

The heat shock transcription factor (HSF) family is involved in regulating growth, development, and abiotic stress. The characteristics and biological functions of HSF family member in X. sorbifolium, an important oil and ornamental plant, have never been reported. In this study, 21 XsHSF genes were identified from the genome of X. sorbifolium and named XsHSF1-XsHSF21 based on their chromosomal positions. Those genes were divided into three groups, A, B, and C, containing 12, one, and eight genes, respectively. Among them, 20 XsHSF genes are located on 11 chromosomes. Protein structure analysis suggested that XsHSF proteins were conserved, displaying typical DNA binding domains (DBD) and oligomerization domains (OD). Moreover, HSF proteins within the same group contain specific motifs, such as motif 5 in the HSFC group. All XsHSF genes have one intron in the CDS region, except XsHSF1 which has two introns. Promoter analysis revealed that in addition to defense and stress responsiveness elements, some promoters also contained a MYB binding site and elements involved in multiple hormones responsiveness and anaerobic induction. Duplication analysis revealed that XsHSF1 and XsHSF4 genes were segmentally duplicated while XsHSF2, XsHSF9, and XsHSF13 genes might have arisen from transposition. Expression pattern analysis of leaves and roots following salt-alkali treatment using qRT-PCR indicated that five XsHSF genes were upregulated and one XsHSF gene was downregulated in leaves upon NaCl treatment suggesting these genes may play important roles in salt response. Additionally, the expression levels of most XsHSFs were decreased in leaves and roots following alkali-induced stress, indicating that those XsHSFs may function as negative regulators in alkali tolerance. MicroRNA target site prediction indicated that 16 of the XsHSF genes may be regulated by multiple microRNAs, for example XsHSF2 might be regulated by miR156, miR394, miR395, miR408, miR7129, and miR854. And miR164 may effect the mRNA levels of XsHSF3 and XsHSF17, XsHSF9 gene may be regulated by miR172. The expression trends of miR172 and miR164 in leaves and roots on salt treatments were opposite to the expression trend of XsHSF9 and XsHSF3 genes, respectively. Promoter analysis showed that XsHSFs might be involved in light and hormone responses, plant development, as well as abiotic stress responses. Our results thus provide an overview of the HSF family in X. sorbifolium and lay a foundation for future functional studies to reveal its roles in saline-alkali response.

Transcriptome analysis of the genes regulating phytohormone and cellular patterning in Lagerstroemia plant architecture.

Plant architecture is a popular research topic because plants with different growth habits that may generate economic or ornamental value are in great demand by orchards and nurseries. However, the molecular basis of the architecture of woody perennial plants is poorly understood due to the complexity of the phenotypic and regulatory relationships. Here, transcriptional profiling of dwarf and non-dwarf crapemyrtles was performed, and potential target genes were identified based on the phenotype, histology and phytohormone metabolite levels. An integrated analysis demonstrated that the internode length was explained mainly by cell number and secondarily by cell length and revealed important hormones in regulatory pathway of Lagerstroemia architecture. Differentially expressed genes (DEGs) involved in phytohormone pathways and cellular patterning regulation were analysed, and the regulatory relationships between these parameters were evaluated at the transcriptional level. Exogenous indole-3-acetic acid (IAA) and gibberellin A4 (GA4) treatments further indicated the pivotal role of auxin in cell division within the shoot apical meristem (SAM) and suggested an interaction between auxin and GA4 in regulating the internode length of Lagerstroemia. These results provide insights for further functional genomic studies on the regulatory mechanisms underlying Lagerstroemia plant architecture and may improve the efficiency of woody plant molecular breeding.

Reference gene selection for qRT-PCR analysis of flower development in Lagerstroemia indica and L. speciosa.

Quantitative real-time polymerase chain reaction (qRT-PCR) is a prevalent method for gene expression analysis, depending on the stability of the reference genes for data normalization. Lagerstroemia indica and L. speciosa are popular ornamental plants which are famous for the long flowering period. However, no systematic studies on reference genes in Lagerstroemia have yet been conducted. In the present study, we selected nine candidate reference genes (GAPDH, TUA, TUB, 18S, RPII, EF-1α, ATC, EIF5A and CYP) and evaluated their expression stability in different tissues during floral development of L. indica and L. speciosa using four algorithms (geNorm, NormFinder, BestKeeper and, RefFinder). Results showed that RPII and EF-1α were the most stably expressed and suitable reference genes for both of Lagerstroemia species. Moreover, ACT exhibited high expression stability in L. indica and GAPDH was a suitable reference gene for L. speciosa in different flower development stages. TUB was an unsuitable reference gene for gene expression normalization due to significant variations in expression across all samples. Finally, we verified the reliability of the selected candidate reference genes by amplifying an AGAMOUS homolog (LsAG1) of Arabidopsis thaliana. This study provides a list of suitable reference genes, thereby broadening the genetic basis of the gene expression patterns in Lagerstroemia species.

Identification and Validation of SNP Markers Linked to Dwarf Traits Using SLAF-Seq Technology in Lagerstroemia.

The genetic control of plant architecture is a promising approach to breed desirable cultivars, particularly in ornamental flowers. In this study, the F1 population (142 seedlings) derived from Lagerstroemia fauriei (non-dwarf) × L. indica 'Pocomoke' (dwarf) was phenotyped for six traits (plant height (PH), internode length (IL), internode number, primary lateral branch height (PLBH), secondary lateral branch height and primary branch number), and the IL and PLBH traits were positively correlated with the PH trait and considered representative indexes of PH. Fifty non-dwarf and dwarf seedlings were pooled and subjected to a specific-locus amplified fragment sequencing (SLAF-seq) method, which screened 1221 polymorphic markers. A total of 3 markers segregating between bulks were validated in the F1 population, with the M16337 and M38412 markers highly correlated with the IL trait and the M25207 marker highly correlated with the PLBH trait. These markers provide a predictability of approximately 80% using a single marker (M25207) and a predictability of 90% using marker combinations (M16337 + M25207) in the F1 population, which revealed that the IL and the PLBH traits, especially the PLBH, were the decisive elements for PH in terms of molecular regulation. Further validation was performed in the BC1 population and a set of 28 Lagerstroemia stocks using allele-specific PCR (AS-PCR) technology, and the results showed the stability and reliability of the SNP markers and the co-determination of PH by multiple genes. Our findings provide an important theoretical and practical basis for the early prediction and indirect selection of PH using the IL and the PLBH, and the detected SNPs may be useful for marker-assisted selection (MAS) in crape myrtle.