Candidate reference genes for quantitative gene expression analysis in Lagerstroemia indica.

Quantitative gene expression analysis by qPCR requires reference genes for normalization. Lagerstroemia indica (crape myrtle) is a popular ornamental plant in the world, but suitable endogenous reference genes are lacking. To find suitable reference genes, we evaluated the stabilities of nine candidate genes in six experimental data sets: six different tissues, three leaf colors, nine flower colors, and under three abiotic stresses (salt, drought, cold) using four statistical algorithms. A target gene LiMYB56 (homolog of Arabidopsis MYB56) was used to verify the authenticity and accuracy of the candidate reference genes. The results showed that the combination of two stably expressed reference genes, rather than a single reference gene, improved the accuracy of the qPCR. LiEF1α-2 + LiEF1α-3 was best for the tissue, salt treatment, and drought treatment sets; LiEF1α-2 + LiEF1α-1 was optimal for leaf color; LiEF1α-2 + LiACT7 was optimal for cold treatment; and LiUBC + LiEF1α-1 was best for the flower color set. Notably, LiEF1α-2 had high expression stability in all six experimental sets, implying it may be a good reference gene for expression studies in L. indica. Our results will facilitate future gene expression studies in L. indica.

GhVLN4 is involved in cell elongation via regulation of actin organization.

MAIN CONCLUSION: GhVLN4 exhibited activity of cross-linking actin filaments into bundles. Overexpression of GhVLN4 increased the abundance of thick actin bundles and resulted in longer cell phenotypes. Actin bundle is a dynamic, higher-order cytoskeleton structure that is essential for cell expansion. Villin is one of the major proteins responsible for crosslinking actin filaments into bundles. However, this kind of actin binding protein has rarely been investigated in cotton. In the present work, a cotton villin gene was molecularly cloned from Upland cotton and denominated as GhVLN4. This gene was more highly expressed in fiber-bearing wild-type cotton TM-1 (Texas Marker-1) than in Ligon lintless-1 mutant (Li-1). Biochemical analysis combined with subcellular localization revealed that GhVLN4 is an actin-binding protein performing actin filament bundling activity in vitro. In line with these findings, a greater abundance of thick actin filament bundles were observed in GhVLN4-overexpressing transgenic plants compared with those in wild-type control. Moreover, ectopic expression of GhVLN4 significantly enhanced the cell length-width ratio of Schizosaccharomyces pombe yeast and increased the length of various Arabidopsis cells, including root cells, root hairs and pollen tubes. Taken together, our results demonstrate that GhVLN4 is involved in the generation of actin filament bundles, suggesting that GhVLN4 may play important roles in regulating plant cell morphogenesis and expansion by remodeling actin cytoskeleton.

Functional divergence of GhCFE5 homoeologs revealed in cotton fiber and Arabidopsis root cell development.

KEY MESSAGE: In GhCFE5 homoeologs, GhCFE5D interacted with more actin homologs and stronger interaction activity than GhCFE5A. GhCFE5D - but not GhCFE5A -overexpression severely disrupted actin cytoskeleton organization and significantly suppressed cell elongation. Homoeologous genes are common in polyploid plants; however, their functional divergence is poorly elucidated. Allotetraploid Upland cotton (Gossypium hirsutum, AADD) is the most widely cultivated cotton; accounting for more than 90 % of the world's cotton production. Here, we characterized GhCFE5A and GhCFE5D homoeologs from G. hirsutum acc TM-1. GhCFE5 homoeologs are expressed preferentially in fiber cells; and a significantly greater accumulation of GhCFE5A mRNA than GhCFE5D mRNA was found in all tested tissues. Overexpression of GhCFE5D but not GhCFE5A seriously inhibits the Arabidopsis hypocotyl and root cell elongation. Yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) analysis showed that compared with GhCFE5A, GhCFE5D interacts with more actin homologs and has a stronger interaction activity both from Arabidopsis and Upland cotton. Interestingly, subcellular localization showed that GhCFE5 resides on the cortical endoplasmic reticulum (ER) network and is colocalized with actin cables. The interaction activities between GhCFE5 homoeologs and actin differ in their effects on F-actin structure in transgenic Arabidopsis root cells. The F-actin changed direction from vertical to lateral, and the actin cytoskeleton organization was severely disrupted in GhCFE5D-overexpressing root cells. These data support the functional divergence of GhCFE5 homoeologs in the actin cytoskeleton structure and cell elongation, implying an important role for GhCFE5 in the evolution and selection of cotton fiber.

GhCFE1A, a dynamic linker between the ER network and actin cytoskeleton, plays an important role in cotton fibre cell initiation and elongation.

Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development.

Phenolic Compounds and Antioxidant Capacity Comparison of Wild-Type and Yellow-Leaf gl1 Mutant of Lagerstroemia indica.

BACKGROUND: The yellow-leaf gl1 mutant of Lagerstroemia indica exhibits an altered phenylpropanoid metabolism pathway compared to wild-type (WT). However, details on the metabolites associated with leaf color variation, including color-specific metabolites with bioactive constituents, are not fully understood. METHODS: Chemical and metabolomics approaches were used to compare metabolite composition and antioxidant capacity between the gl1 mutant and WT leaves. RESULTS: The mutant exhibited an irregular xylem structure with a significantly lower phenolic polymer lignin content and higher soluble phenolic compounds. Untargeted metabolomics analysis identified phenolic compounds, particularly lignans, as key differential metabolites between gl1 and WT, with a significant increase in the mutant. The neolignan derivative balanophonin-4-O-D-glu was identified as a characteristic metabolite in the gl1 mutant. The soluble phenolic compounds of the gl1 mutant exhibited higher FRAP, ABTS, DPPH, and hydroxyl radical scavenging activity than in WT. Correlation analysis showed a positive relationship between antioxidant capacity and phenolic compounds in L. indica. CONCLUSIONS: Metabolites associated with leaf color variation in the L. indica yellow-leaf gl1 mutant demonstrated high antioxidant capacity, particularly in scavenging hydroxyl radicals.

Discovery and identification of a novel Ligon lintless-like mutant (Lix) similar to the Ligon lintless (Li1) in allotetraploid cotton.

Mutants are a powerful resource for studying gene structure, function, and evolution. In this present study, a novel Ligon lintless-like mutant (Lix), that has short fibers and deformed leaves and stems, was isolated from the progeny of transgenic cottons. The Lix mutant is similar in morphology to the Ligon lintless (Li1) mutant. Genetic analysis and molecular mapping were performed for the Lix and Li1 mutants. These two mutants are monogenic dominant mutants with distorted growth of vegetative and reproductive structures. Seedlings of the dominant homozygote Li 1 Li 1 genotype are lethal, while LixLix plants are viable but show no reproductive growth. Molecular tagging showed that the Lix gene is located on Chr. 04 in a 30.9-cM region spanned by NAU8376 and NAU3469. In a previous study, the Li 1 gene was mapped to Chr. 22, and Chr. 04 and Chr. 22 are homoelogous chromosomes in tetraploid cotton. So, we propose that Lix and Li1 mutants have similar mutated morphology, and Lix is mapped to a homoelogous chromosome carrying Li 1 . The identification and genetic mapping of Lix/Li 1 genes using mutants provides a foundation for isolating these genes. In turn, this will permit studies to elucidate the functional and evolutionary roles for these genes in cotton growth and development.

The oxidative damage of the Lagerstroemia indica chlorosis mutant gl1 involves in ferroptosis.

Photooxidation is the major physiological performance of the Lagerstroemia indica chlorosis mutant gl1 under field conditions. The mechanisms of the progressive symptoms of oxidative damage from the lower older leaves to the upper mature leaves are complicated and still unclear. The aim of this work was to investigate the physiological mechanisms of oxidative stress from the perspective of the photosynthetic metabolites. The phytosynthetic metabolites of gl1 mutant changed significantly compared to wild type (WT) L. indica, such as by increasing phenolics, decreasing soluble sugar, protein and ascorbate, and redistributing antioxidant enzyme activities. The co-accumulation of phenolics and guaiacol-POD in gl1 mutant promote the removal of H2O2, as well the increase of phenoxyl radicals levels. Furthermore, the ion balance was significantly disturbed and Fe accumulated the most among these fluctuating nutrients in the leaves of gl1 mutant. The accumulated Fe was found neither in the chloroplasts nor in the cell wall of the leaves and became unshielded Fe, which favors the Fenton/Haber-Weiss reaction and stabilizes the phenoxyl radicals in metal complexation. The results suggested that the increase of phenolics and Fe accumulation were obviously involved in oxidative damage of gl1 mutant.

GhVLN4 is involved in multiple stress responses and required for resistance to Verticillium wilt.

As structural and signaling platform in plant cell, the actin cytoskeleton is regulated by diverse actin binding proteins (ABPs). Villins are one type of major ABPs responsible for microfilament bundling, which have proved to play important roles in plant growth and development. However, the function of villins in stress tolerance is poorly understood. Here, we report the function of cotton GhVLN4 in Verticillium wilt resistance and abiotic stress tolerance. The expression of GhVLN4 was up-regulated by gibberellin, ethylene, ABA, salicylic acid, jasmonate, NaCl, PEG, and Verticillium dahliae treatment, suggesting the involvement of GhVLN4 in multiple stress and hormone responses and signaling. Virus-induced gene silencing GhVLN4 made cotton more susceptible to V. dahliae characterized by the preferential colonization and rapid growth of the fungus in both phloem and xylem of the infected stems. Arabidopsis overexpressing GhVLN4 exhibited higher resistance to V. dahliae, salt and drought than the wild-type plants. The enhanced resistance to V. dahliae is likely related to the upregulated components in SA signaling pathway; the improved tolerance to salt and drought is characterized by upregulation of the components both in ABA- related and ABA-independent signal pathways, along with altered stomatal aperture under drought. Our findings demonstrate that GhVLN4 may play important roles in regulating plant tolerance to both biotic and abiotic stresses.

Efficient Transformation of Catalpa bungei Shows Crystal Genes Conferring Resistance to the Shoot Borer Omphisa plagialis.

Although Catalpa bungei is a forest plant with considerable economic and ornamental value in China, its wood and decorative qualities are constrained by insect pests such as the shoot borer Omphisa plagialis (Lepidoptera). Overexpressing insect resistance genes such as crystal genes to develop an insect-resistant variety of C. bungei is an environmental and ecological approach. However, genotype limitations and low regeneration rates of embryogenic calli (EC) inhibit the development of transformation and the insect-resistant gene expression system in C. bungei. Here, we first established embryogenic callus induction and regeneration systems of five genotypes using mature seed and stem segment explants; the highest induction and regeneration rates of EC were 39.89 and 100%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was developed from EC and its positive frequency was up to 92.31%. Finally, using the transformation system, 15 and 22 transgenic C. bungei lines that expressed Cry2A and Cry9Aa-like were generated, respectively. These transgenic lines that exhibited significantly higher resistance to O. plagialis in the laboratory and field have great promise for meeting the challenge of future pest management under changing climatic conditions. Additionally, this efficient, fast, and stable transformation system could be a potential tool for gene function analysis and forest tree genetic improvement.

The essential role of GhPEL gene, encoding a pectate lyase, in cell wall loosening by depolymerization of the de-esterified pectin during fiber elongation in cotton.

Cotton fiber elongation, largely achieved by cell wall loosening, is an important stage during cotton fiber development. In this present research, a fiber preferential cDNA encoding a pectate lyase (PEL) which could exclusively degrade the de-esterified pectin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. Subsequently, the corresponding PEL genes were isolated from four different cotton species and characterized. In vitro enzyme assays indicated that GhPEL really exhibited cleavage-activity against de-esterified pectin. The temporal-spatial expression analyses revealed that the GhPEL gene was preferentially expressed in fibers at 10 days-post anthesis (DPA). Antisense GhPEL transgenic cotton plants were generated by Agrobacterium-mediated transformation. Six homozygous lines, each with one or two copies of the transgene inserted as determined by southern blot analysis of the NPTII gene, were selected for further functional analysis. The GhPEL expression during fiber elongation in these transgenic lines was significantly suppressed in various degrees. Furthermore, the reduction of GhPEL enzymatic activity by decreasing GhPEL transcripts severely affected the degradation of de-esterified pectin in primary cell walls of transgenic cotton fibers, which consequently blocked cell wall loosening in early fiber development. Ultimately, the fiber elongation of all these transgenic lines was repressed. These results suggested that GhPEL may play an important role in the process of normal fiber elongation in cotton.

Sequence-based ultra-dense genetic and physical maps reveal structural variations of allopolyploid cotton genomes.

BACKGROUND: SNPs are the most abundant polymorphism type, and have been explored in many crop genomic studies, including rice and maize. SNP discovery in allotetraploid cotton genomes has lagged behind that of other crops due to their complexity and polyploidy. In this study, genome-wide SNPs are detected systematically using next-generation sequencing and efficient SNP genotyping methods, and used to construct a linkage map and characterize the structural variations in polyploid cotton genomes. RESULTS: We construct an ultra-dense inter-specific genetic map comprising 4,999,048 SNP loci distributed unevenly in 26 allotetraploid cotton linkage groups and covering 4,042 cM. The map is used to order tetraploid cotton genome scaffolds for accurate assembly of G. hirsutum acc. TM-1. Recombination rates and hotspots are identified across the cotton genome by comparing the assembled draft sequence and the genetic map. Using this map, genome rearrangements and centromeric regions are identified in tetraploid cotton by combining information from the publicly-available G. raimondii genome with fluorescent in situ hybridization analysis. CONCLUSIONS: We report the genotype-by-sequencing method used to identify millions of SNPs between G. hirsutum and G. barbadense. We construct and use an ultra-dense SNP map to correct sequence mis-assemblies, merge scaffolds into pseudomolecules corresponding to chromosomes, detect genome rearrangements, and identify centromeric regions in allotetraploid cottons. We find that the centromeric retro-element sequence of tetraploid cotton derived from the D subgenome progenitor might have invaded the A subgenome centromeres after allotetrapolyploid formation. This study serves as a valuable genomic resource for genetic research and breeding of cotton.