The complex genome and adaptive evolution of polyploid Chinese pepper (Zanthoxylum armatum and Zanthoxylum bungeanum)

Zanthoxylum armatum and Zanthoxylum bungeanum, known as ‘Chinese pepper’, are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species-special metabolites. Here, we report reference-grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole-genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high-quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes.     

High-temperature stress in crops: male sterility,yield loss and potential remedy approaches

Global food security is one of the utmost essential challenges in the 21st century in providing enough food for the growing population while coping with the already stressed environment. High temperature (HT) is one of the main factors affecting plant growth, development and reproduction and causes male sterility in plants. In male reproductive tissues, metabolic changes induced by HT involve carbohydrates, lipids, hormones, epigenetics and reactive oxygen species, leading to male sterility and ultimately reducing yield. Understanding the mechanism and genes involved in these pathways during the HT stress response will provide a new path to improve crops by using molecular breeding and biotechnological approaches. Moreover, this review provides insight into male sterility and integrates this with suggested strategies to enhance crop tolerance under HT stress conditions at the reproductive stage.     

Expression of Fungal Cutinase and Swollenin in Tobacco Chloroplasts Reveals Novel Enzyme Functions and/or Substrates

In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis.     

Production and characterization of somatic hybrids between upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) and wild cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> Anderss) via electrofusion

Symmetric somatic hybrid plants between Gossypium hirsutum Coker 201 and G. klotzschianum were obtained through electrofusion. The fusion products were cultured in KM₈P medium supplemented with 2.685 M -naphthaleneacetic acid and 0.465 M kinetin, and the regenerated plants were morphologically, genetically, and cytologically characterized. Nuclear-DNA flow cytometric analysis revealed that the plants tested (31 of 40) had a relative DNA content close to the total DNA contents of the two parents. Subsequent genome DNA analysis using random amplified polymorphic DNA markers revealed 16 of 18 plants were true somatic hybrids. Cytological investigation of the metaphase root-tip cells of seven hybrids revealed there were 72–81 chromosomes in the hybrids, a value close to the expected 78 chromosomes. The morphology of the hybrids was distinct from that of the parents and from that of the regenerants from protoplasts of Coker 201. Somatic hybridization represents a potent and novel tool for transferring genomes of wild cottons containing economically important traits to cultivars in breeding programs. This is the first report on the regeneration of somatic hybrids via protoplast fusion in cotton.     

Chromatin reorganization and endogenous auxin/cytokinin dynamic activity during somatic embryogenesis of cultured cotton cell

We conducted a systematic assessment and comparative study on the biochemical and cellular characteristics of cultured cotton cells during the entire process of somatic embryogenesis (SE). All staged cultures were widely investigated in this assay. Cell and tissue ectogenesis manipulation combined with flow cytometry (FCM) was employed to cellular study during the whole totipotency process of dedifferentiation and redifferentiation. We identified two phases of chromatin decondensation during the dedifferentiation and redifferentiation. At the same time, sharp increase in the ratio of indoleacetic acid (IAA), isopentenyladenosine group (iPAs) at the same stage of cell dedifferentiation and redifferentiation process serve as distinct biochemical maker of dedifferentiation and SE initiation with the unique feature. Our results suggest the two phases of chromatin reorganization associated with endogenous auxin/cytokinin dynamic activity may underlie dedifferentiation and redifferentiation during the entire SE process in cotton.     

Factors affecting transformation efficiency of embryogenic callus of Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) with <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 10⁸ cells ml^–1 (OD₆₀₀=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l^–1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.     

Stevioside ameliorates high-fat diet-induced insulin resistance and adipose tissue inflammation by downregulating the NF-κB pathway

Accumulating evidence suggests that adipose tissue is the main source of pro-inflammatory molecules that predispose individuals to insulin resistance. Stevioside (SVS) is a widely used sweetener with multiple beneficial effects for diabetic patients. In this study, we investigated the effect of SVS on insulin resistance and the pro-inflammatory state of adipose tissue in mice fed with a high-fat diet (HFD). Oral administration of SVS for 1month had no effect on body weight, but it significantly improved fasting glucose, basal insulin levels, glucose tolerance and whole body insulin sensitivity. Interestingly, these changes were accompanied with decreased expression levels of several inflammatory cytokines in adipose tissue, including TNF-α, IL6, IL10, IL1β, KC, MIP-1α, CD11b and CD14. Moreover, macrophage infiltration in adipose tissue was remarkably reduced by SVS. Finally, SVS significantly suppressed the nuclear factor-kappa b (NF-κB) signaling pathway in adipose tissue. Collectively, these results suggested that SVS may ameliorate insulin resistance in HFD-fed mice by attenuating adipose tissue inflammation and inhibiting the NF-κB pathway.     

High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.