Starch and sucrose metabolism plays an important role in the stem development in Medicago sativa.

The forage quality of alfalfa (Medicago sativa ) stems is greater than the leaves. Sucrose hydrolysis provides energy for stem development, with starch being enzymatically converted into sucrose to maintain energy homeostasis. To understand the physiological and molecular networks controlling stem development, morphological characteristics and transcriptome profiles in the stems of two alfalfa cultivars (Zhungeer and WL168) were investigated. Based on transcriptome data, we analysed starch and sugar contents, and enzyme activity related to starch-sugar interconversion. Zhungeer stems were shorter and sturdier than WL168, resulting in significantly higher mechanical strength. Transcriptome analysis showed that starch and sucrose metabolism were significant enriched in the differentially expressed genes of stems development in both cultivars. Genes encoding INV , bglX , HK , TPS and glgC downregulated with the development of stems, while the gene encoding was AMY upregulated. Weighted gene co-expression network analysis revealed that the gene encoding glgC was pivotal in determining the variations in starch and sucrose contents between the two cultivars. Soluble carbohydrate, sucrose, and starch content of WL168 were higher than Zhungeer. Enzyme activities related to sucrose synthesis and hydrolysis (INV, bglX, HK, TPS) showed a downward trend. The change trend of enzyme activity was consistent with gene expression. WL168 stems had higher carbohydrate content than Zhungeer, which accounted for more rapid growth and taller plants. WL168 formed hollow stems were formed during rapid growth, which may be related to the redistribution of carbohydrates in the pith tissue. These results indicated that starch and sucrose metabolism play important roles in the stem development in alfalfa.

Climate of origin shapes variations in wood anatomical properties of 17 Picea species.

BACKGROUND: Variations in hydraulic conductivity may arise from species-specific differences in the anatomical structure and function of the xylem, reflecting a spectrum of plant strategies along a slow-fast resource economy continuum. Spruce (Picea spp.), a widely distributed and highly adaptable tree species, is crucial in preventing soil erosion and enabling climate regulation. However, a comprehensive understanding of the variability in anatomical traits of stems and their underlying drivers in the Picea genus is currently lacking especially in a common garden. RESULTS: We assessed 19 stem economic properties and hydraulic characteristics of 17 Picea species grown in a common garden in Tianshui, Gansu Province, China. Significant interspecific differences in growth and anatomical characteristics were observed among the species. Specifically, xylem hydraulic conductivity (Ks) and hydraulic diameter exhibited a significant negative correlation with the thickness to span ratio (TSR), cell wall ratio, and tracheid density and a significant positive correlation with fiber length, and size of the radial tracheid. PCA revealed that the first two axes accounted for 64.40% of the variance, with PC1 reflecting the trade-off between hydraulic efficiency and mechanical support and PC2 representing the trade-off between high embolism resistance and strong pit flexibility. Regression analysis and structural equation modelling further confirmed that tracheid size positively influenced Ks, whereas the traits DWT, D_r, and TSR have influenced Ks indirectly. All traits failed to show significant phylogenetic associations. Pearson's correlation analysis demonstrated strong correlations between most traits and longitude, with the notable influence of the mean temperature during the driest quarter, annual precipitation, precipitation during the wettest quarter, and aridity index. CONCLUSIONS: Our results showed that xylem anatomical traits demonstrated considerable variability across phylogenies, consistent with the pattern of parallel sympatric radiation evolution and global diversity in spruce. By integrating the anatomical structure of the stem xylem as well as environmental factors of origin and evolutionary relationships, our findings provide novel insights into the ecological adaptations of the Picea genus.

High-density genetic map construction and QTL mapping of a zigzag-shaped stem trait in tea plant (Camellia sinensis).

The highly unique zigzag-shaped stem phenotype in tea plants boasts significant ornamental value and is exceptionally rare. To investigate the genetic mechanism behind this trait, we developed BC1 artificial hybrid populations. Our genetic analysis revealed the zigzag-shaped trait as a qualitative trait. Utilizing whole-genome resequencing, we constructed a high-density genetic map from the BC1 population, incorporating 5,250 SNP markers across 15 linkage groups, covering 3,328.51 cM with an average marker interval distance of 0.68 cM. A quantitative trait locus (QTL) for the zigzag-shaped trait was identified on chromosome 4, within a 61.2 to 97.2 Mb range, accounting for a phenotypic variation explained (PVE) value of 13.62%. Within this QTL, six candidate genes were pinpointed. To better understand their roles, we analyzed gene expression in various tissues and individuals with erect and zigzag-shaped stems. The results implicated CsXTH (CSS0035625) and CsCIPK14 (CSS0044366) as potential key contributors to the zigzag-shaped stem formation. These discoveries lay a robust foundation for future functional genetic mapping and tea plant genetic enhancement.

Parametrization of biological assumptions to simulate growth of tree branching architectures.

Modeling and simulating the growth of the branching of tree species remains a challenge. With existing approaches, we can reconstruct or rebuild the branching architectures of real tree species, but the simulation of the growth process remains unresolved. First, we present a tree growth model to generate branching architectures that resemble real tree species. Secondly, we use a quantitative morphometric approach to infer the shape similarity of the generated simulations and real tree species. Within a functional-structural plant model, we implement a set of biological parameters that affect the branching architecture of trees. By modifying the parameter values, we aim to generate basic shapes of spruce, pine, oak and poplar. Tree shapes are compared using geometric morphometrics of landmarks that capture crown and stem outline shapes. Five biological parameters, namely xylem flow, shedding rate, proprioception, gravitysense and lightsense, most influenced the generated tree branching patterns. Adjusting these five parameters resulted in the different tree shapes of spruce, pine, oak, and poplar. The largest effect was attributed to gravity, as phenotypic responses to this effect resulted in different growth directions of gymnosperm and angiosperm branching architectures. Since we were able to obtain branching architectures that resemble real tree species by adjusting only a few biological parameters, our model is extendable to other tree species. Furthermore, the model will also allow the simulation of structural tree-environment interactions. Our simplifying approach to shape comparison between tree species, landmark geometric morphometrics, showed that even the crown-trunk outlines capture species differences based on their contrasting branching architectures.

Adaptation mechanism of three Impatiens species to different habitats based on stem morphology, lignin and MYB4 gene.

BACKGROUND: Impatiens is an important genus with rich species of garden plants, and its distribution is extremely extensive, which is reflected in its diverse ecological environment. However, the specific mechanisms of Impatiens' adaptation to various environments and the mechanism related to lignin remain unclear. RESULTS: Three representative Impatiens species,Impatiens chlorosepala (wet, low degree of lignification), Impatiens uliginosa (aquatic, moderate degree of lignification) and Impatiens rubrostriata (terrestrial, high degree of lignification), were selected and analyzed for their anatomical structures, lignin content and composition, and lignin-related gene expression. There are significant differences in anatomical parameters among the stems of three Impatiens species, and the anatomical structure is consistent with the determination results of lignin content. Furthermore, the thickness of the xylem and cell walls, as well as the ratio of cell wall thickness to stem diameter have a strong correlation with lignin content. The anatomical structure and degree of lignification in Impatiens can be attributed to the plant's growth environment, morphology, and growth rate. Our analysis of lignin-related genes revealed a negative correlation between the MYB4 gene and lignin content. The MYB4 gene may control the lignin synthesis in Impatiens by controlling the structural genes involved in the lignin synthesis pathway, such as HCT, C3H, and COMT. Nonetheless, the regulation pathway differs between species of Impatiens. CONCLUSIONS: This study demonstrated consistency between the stem anatomy of Impatiens and the results obtained from lignin content and composition analyses. It is speculated that MYB4 negatively regulates the lignin synthesis in the stems of three Impatiens species by regulating the expression of structural genes, and its regulation mechanism appears to vary across different Impatiens species. This study analyses the variations among different Impatiens plants in diverse habitats, and can guide further molecular investigations of lignin biosynthesis in Impatiens.

Stem moisture content prediction model for Larix olgensis based on beta regression.

To investigate the longitudinal variation patterns of sapwood, heartwood, bark and stem moisture content along the trunk of artificial Larix olgensis, we constructed mixed effect models of moisture content based on beta regression by combining the effects of sampling plot and sample trees. We used two sampling schemes to calibrate the model, without limiting the relative height (Scheme Ⅰ) and with a limiting height of less than 2 m (Scheme II). The results showed that sapwood and stem moisture content increased gradually along the trunk, heartwood moisture content decreased slightly and then increased along the trunk, and bark moisture content increased along the trunk and then levelled off before increasing. Relative height, height to crown base, stand area at breast height per hectare, age, and stand dominant height were main factors driving moisture content of L. olgensis. Scheme Ⅰ showed the stable prediction accuracy when randomly sampling moisture content measurements from 2-3 discs to calibrate the model, with the mean absolute percentage error (MAPE) of up to 7.2% for stem moisture content (randomly selected 2 discs), and the MAPE of up to 7.4%, 10.5% and 10.5% for sapwood, heartwood and bark moisture content (randomly selected 3 discs), respectively. Scheme Ⅱ was appropriate when sampling moisture content measurements from discs of 1.3 and 2 m height and the MAPE of sapwood, heartwood, bark and stem moisture content reached 7.8%, 11.0%, 10.4% and 7.1%, respectively. The prediction accuracies of all mixed effect beta regression models were better than the base model. The two-level mixed effect beta regression models, considering both plot effect and tree effect, would be suitable for predicting moisture content of each part of L. olgensis well.

Identification and characterization of PAL genes involved in the regulation of stem development in Saccharum spontaneum L.

BACKGROUND: Saccharum spontaneum L. is a closely related species of sugarcane and has become an important genetic component of modern sugarcane cultivars. Stem development is one of the important factors for affecting the yield, while the molecular mechanism of stem development remains poorly understanding in S. spontaneum. Phenylalanine ammonia-lyase (PAL) is a vital component of both primary and secondary metabolism, contributing significantly to plant growth, development and stress defense. However, the current knowledge about PAL genes in S. spontaneum is still limited. Thus, identification and characterization of the PAL genes by transcriptome analysis will provide a theoretical basis for further investigation of the function of PAL gene in sugarcane. RESULTS: In this study, 42 of PAL genes were identified, including 26 SsPAL genes from S. spontaneum, 8 ShPAL genes from sugarcane cultivar R570, and 8 SbPAL genes from sorghum. Phylogenetic analysis showed that SsPAL genes were divided into three groups, potentially influenced by long-term natural selection. Notably, 20 SsPAL genes were existed on chromosomes 4 and 5, indicating that they are highly conserved in S. spontaneum. This conservation is likely a result of the prevalence of whole-genome replications within this gene family. The upstream sequence of PAL genes were found to contain conserved cis-acting elements such as G-box and SP1, GT1-motif and CAT-box, which collectively regulate the growth and development of S. spontaneum. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that SsPAL genes of stem had a significantly upregulated than that of leaves, suggesting that they may promote the stem growth and development, particularly in the + 6 stem (The sixth cane stalk from the top to down) during the growth stage. CONCLUSIONS: The results of this study revealed the molecular characteristics of SsPAL genes and indicated that they may play a vital role in stem growth and development of S. spontaneum. Altogether, our findings will promote the understanding of the molecular mechanism of S. spontaneum stem development, and also contribute to the sugarcane genetic improving.

Positive contributions of the stem to the formation of white tea quality-related metabolites during withering.

Most teas, including white tea, are produced from tender shoots containing both leaf and stem. However, the effect of the stem on white tea quality remains unclear, especially during withering, an essential process. Therefore, this study investigated the withering-induced changes in the leaves and stems of Camellia sinensis cv. 'Fudingdabai' by multi-group analysis. During withering, the levels of catechin and theobromine (i.e., major flavor-related compounds) decreased slightly, mainly in the leaves. The abundance of some proteinaceous amino acids related to fresh taste increased in stems due to increased protein hydrolysis. In addition, changes in biosynthetic pathways caused a decrease in theanine (a major non-proteinaceous amino acid) and an increase in gamma-aminobutyric acid in stems. Terpenes, mainly in the stems, were partially affected by withering. Phenylacetaldehyde, a major contributor to white tea aroma, increased mainly in the stems. These findings reflect the positive contribution of the stem to white tea quality.

Blue light regulated lignin and cellulose content of soybean petioles and stems under low light intensity.

To improve light harvest and plant structural support under low light intensity, it is useful to investigate the effects of different ratios of blue light on petiole and stem growth. Two true leaves of soybean seedlings were exposed to a total light intensity of 200µmolm-2 s-1 , presented as either white light or three levels of blue light (40µmolm-2 s-1 , 67µmolm-2 s-1 and 100µmolm-2 s-1 ) for 15days. Soybean petioles under the low blue light treatment upregulated expression of genes relating to lignin metabolism, enhancing lignin content compared with the white light treatment. The low blue light treatment had high petiole length, increased plant height and improved petiole strength arising from high lignin content, thus significantly increasing leaf dry weight relative to the white light treatment. Compared with white light, the treatment with the highest blue light ratio reduced plant height and enhanced plant support through increased cellulose and hemicellulose content in the stem. Under low light intensity, 20% blue light enhanced petiole length and strength to improve photosynthate biomass; whereas 50% blue light lowered plants' centre of gravity, preventing lodging and conserving carbohydrate allocation.

Temperate woody species across the angiosperm phylogeny acquire tolerance to water deficit stress during the growing season.

Understanding the capacity of temperate trees to acclimate to limited soil water has become essential in the face of increasing drought risk due to climate change. We documented seasonal - or phenological - patterns in acclimation to water deficit stress in stems and leaves of tree species spanning the angiosperm phylogeny. Over 3 yr of field observations carried out in two US arboreta, we measured stem vulnerability to embolism (36 individuals of 7 Species) and turgor loss point (119 individuals of 27 species) over the growing season. We also conducted a growth chamber experiment on 20 individuals of one species to assess the mechanistic relationship between soil water restriction and acclimation. In three-quarters of species measured, plants became less vulnerable to embolism and/or loss of turgor over the growing season. We were able to stimulate this acclimatory effect by withholding water in the growth chamber experiment. Temperate angiosperms are capable of acclimation to soil water deficit stress, showing maximum vulnerability to soil water deficits following budbreak and becoming more resilient to damage over the course of the growing season or in response to simulated drought. The species-specific tempo and extent of this acclimatory potential constitutes preadaptive climate change resilience.

Multimodal Spectroscopic Studies to Evaluate the Effect of Nod-Factor-Based Fertilizer on the Maize (Zea mays) Stem.

Maize (Zea mays) is one of the most cultivated plants in the world. Due to the large area, the scale of its production, and the demand to increase the yield, there is a need for new environmentally friendly fertilizers. One group of such candidates is bacteria-produced nodulation (or nod) factors. Limited research has explored the impact of nodulation, factors on maize within field conditions, with most studies restricted to greenhouse settings and early developmental stages. Additionally, there is a scarcity of investigations that elucidate the metabolic alterations in the maize stem due to nod-factor exposure. It was therefore the aim of this study. Maize stem's metabolites and fibers were analyzed with various imaging analytical techniques: matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR), and diffuse reflectance infrared Fourier transform spectroscopy. Moreover, the biochemical analyses were used to evaluate the proteins and soluble carbohydrates concentration and total phenolic content. These techniques were used to evaluate the influence of nod factor-based biofertilizer on the growth of a non-symbiotic plant, maize. The biofertilizer increased the grain yield and the stem mass. Moreover, the spectroscopic and biochemical investigation proved the appreciable biochemical changes in the stems of the maize in biofertilizer-treated plants. Noticeable changes were found in the spatial distribution and the increase in the concentration of flavonoids such as maysin, quercetin, and rutin. Moreover, the concentration of cell wall components (fibers) increased. Furthermore, it was shown that the use of untargeted analyses (such as Raman and ATR FT-IR, spectroscopic imaging, and MALDI-MSI) is useful for the investigation of the biochemical changes in plants.

SULTR2;1 Adjusts the Bolting Timing by Transporting Sulfate from Rosette Leaves to the Primary Stem.

Sulfur (S) is an essential macronutrient for plant growth and metabolism. SULTR2;1 is a low-affinity sulfate transporter facilitating the long-distance transport of sulfate in Arabidopsis. The physiological function of SULTR2;1 in the plant life cycle still needs to be determined. Therefore, we analyzed the sulfate transport, S-containing metabolite accumulation and plant growth using Arabidopsis SULTR2;1 disruption lines, sultr2;1-1 and sultr2;1-2, from seedling to mature growth stages to clarify the metabolic and physiological roles of SULTR2;1. We observed that sulfate distribution to the stems was affected in sultr2;1 mutants, resulting in decreased levels of sulfate, cysteine, glutathione (GSH) and total S in the stems, flowers and siliques; however, the GSH levels increased in the rosette leaves. This suggested the essential role of SULTR2;1 in sulfate transport from rosette leaves to the primary stem. In addition, sultr2;1 mutants unexpectedly bolted earlier than the wild-type without affecting the plant biomass. Correlation between GSH levels in rosette leaves and the bolting timing suggested that the rosette leaf GSH levels or limited sulfate transport to the early stem can trigger bolting. Overall, this study demonstrated the critical roles of SULTR2;1 in maintaining the S metabolite levels in the aerial part and transitioning from the vegetative to the reproductive growth phase.

Isohydric stomatal behaviour alters fruit vascular flows and minimizes fruit size reductions in drought-stressed 'Hass' avocado (Persea americana Mill.).

BACKGROUND AND AIMS: Plant water status is important for fruit development, because many fleshy fruits contain large amounts of water. However, there is no information on vascular flows of Persea americana 'Hass' avocado. The aims of this research were to explore the impact of drought stress on the water relationships of the 'Hass' avocado plant and its fruit growth. METHODS: Well-watered and water-stressed 'Hass' avocado plants were compared. Over 4 weeks, water flows through the shoot and fruit pedicel were monitored using external sap flow gauges. Fruit diameter was monitored using linear transducers, and stomatal conductance (gs), photosynthesis (A) and leaf and stem water potentials (Ñ°leaf and Ñ°stem) were measured to assess the response of the plants to water supply. KEY RESULTS: In well-watered conditions, the average water inflow to the shoot was 72 g day-1. Fruit water inflow was 2.72 g day-1, but there was water loss of 0.37 g day-1 caused by the outflow (loss back into the tree) through the vascular tissues and 1.06 g day-1 from the fruit skin. Overall, fruit volume increased by 1.4 cm3 day-1. In contrast, water flow into fruit of water-stressed plants decreased to 1.88 g day-1, with the outflow increasing to 0.61 g day-1. As a result, increases in fruit volume were reduced to 0.4 cm3 day-1. The values of A, gs and sap flow to shoots were also reduced during drought conditions. Changes in the hourly time-courses of pedicel sap flow, fruit volume and stem water potential during drought suggest that the stomatal response prevented larger increases in outflow from the fruit. Following re-watering, a substantial recovery in growth rate was observed. CONCLUSIONS: In summary, a reduction in growth of avocado fruit was observed with induced water deficit, but the isohydric stomatal behaviour of the leaves helped to minimize negative changes in water balance. Also, there was substantial recovery after re-watering, hence the short-term water stress did not decrease avocado fruit size. Negative impacts might appear if the drought treatment were prolonged.

Air spaces bend light in plant stems.

Intercellular air spaces are necessary for phototropism in Arabidopsis thaliana.

Characterization of hemicellulose in Cunninghamia lanceolata stem during xylogenesis.

In this study, hemicellulose was isolated from the apical, middle and basal segments of C. lanceolata stem to investigate the dynamic change of its structure during xylogenesis. Results showed that the C. lanceolata hemicellulose is mainly consisted of O-acetylgalactoglucomannan (GGM) which backbone is alternately linked by ß-d-mannopyranosyl (Manp) and ß-d-glucopyranosyl (Glcp) via (1 â†’ 4)-glycosidic bond, while the side chains are α-d-galactopyranosyl (Galp) and acetyl. In addition, 4-O-methylglucuronoarabinoxylan (GAX) is another dominant structure of C. lanceolata hemicellulose which contains a linear backbone of (1 â†’ 4)-ß-d-xylopyranosyl (Xylp) and side chains of 4-O-Me-α-d-glucuronic acid (MeGlcpA) and α-L-arabinofuranose (Araf). The thickness of the cell wall, the ratio of GGM/GAX and the molecular weight of hemicellulose were increased as the extension of growth time. The degree of glycosyl substitutions of xylan and mannan was decreased from 10.34 % (apical) to 8.38 % (basal) and from 15.63 % (apical) to 10.49 % (basal), respectively. However, the total degree of acetylation was enhanced from 0.28 (apical) to 0.37 (basal). Transcriptome analysis showed that genes (CSLA9, IRX9H1, IRX10L, IRX15L, GMGT1, TBL19, TBL25, GUX2, GUX3, GXM1, F8H1 and F8H2) related to hemicellulose biosynthesis are mainly expressed in mature part. This study is of great significance for genetic breeding and high-value utilization of C. lanceolata.

Warm springs alter timing but not total growth of temperate deciduous trees.

As the climate changes, warmer spring temperatures are causing earlier leaf-out1-3 and commencement of CO2 uptake1,3 in temperate deciduous forests, resulting in a tendency towards increased growing season length3 and annual CO2 uptake1,3-7. However, less is known about how spring temperatures affect tree stem growth8,9, which sequesters carbon in wood that has a long residence time in the ecosystem10,11. Here we show that warmer spring temperatures shifted stem diameter growth of deciduous trees earlier but had no consistent effect on peak growing season length, maximum growth rates, or annual growth, using dendrometer band measurements from 440 trees across two forests. The latter finding was confirmed on the centennial scale by 207 tree-ring chronologies from 108 forests across eastern North America, where annual ring width was far more sensitive to temperatures during the peak growing season than in the spring. These findings imply that any extra CO2 uptake in years with warmer spring temperatures4,5 does not significantly contribute to increased sequestration in long-lived woody stem biomass. Rather, contradicting projections from global carbon cycle models1,12, our empirical results imply that warming spring temperatures are unlikely to increase woody productivity enough to strengthen the long-term CO2 sink of temperate deciduous forests.

Identification of genes associated with biosynthesis of bioactive flavonoids and taxoids in Taxus cuspidata Sieb. et Zucc. plantlets exposed to UV-B radiation.

UV-B radiation is a typical environmental stressor that can promote phytochemical accumulation in plants. Taxus species are highly appreciated due to the existence of bioactive taxoids (especially paclitaxel) and flavonoids. However, the effect of UV-B radiation on taxoid and flavonoid biosynthesis in Taxus cuspidata Sieb. et Zucc. is largely unknown. In the present work, the accumulation of taxoids and flavonoids in T. cuspidata plantlets was significantly induced by 12 and 24 h of UV-B radiation (3 W/m2), and a large number of significantly differentially expressed genes were obtained via transcriptomic analysis. The significant up-regulation of antioxidant enzyme- and flavonoid biosynthesis-related genes (phenylalanine ammonia lyase 1, chalcone synthase 2, flavonol synthase 1, and flavonoid 3', 5'-hydroxylase 2), suggested that UV-B might cause the oxidative stress thus promoting flavonoid accumulation in T. cuspidata. Moreover, the expression of some genes related to jasmonate metabolism and taxoid biosynthesis (taxadiene synthase, baccatin III-3-amino 3-phenylpropanoyltransferase 1, taxadiene-5α-hydroxylase, and ethylene response factors 15) was significantly activated, which indicated that UV-B might initiate jasmonate signaling pathway that contributed to taxoid enhancement in T. cuspidata. Additionally, the identification of some up-regulated genes involved in lignin biosynthesis pathway indicated that the lignification process in T. cuspidata might be stimulated for defense against UV-B radiation. Overall, our findings provided a better understanding of some potential key genes associated with flavonoid and taxoid biosynthesis in T. cuspidata exposed to UV-B radiation.

Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation.

Catharanthus roseus (L.) G. Don is a plant belonging to the genus Catharanthus of the Apocynaceae family. It contains more than one hundred alkaloids, of which some exhibit significant pharmacological activities. Chitooligosaccharides are the only basic aminooligosaccharides with positively charged cations in nature, which can regulate plant growth and antioxidant properties. In this study, the leaves of Catharanthus roseus were sprayed with chitooligosaccharides of different molecular weights (1 kDa, 2 kDa, 3 kDa) and different concentrations (0.01 µg/mL, 0.1 µg/mL, 1 µg/mL and 10 µg/mL). The fresh weights of its root, stem and leaf were all improved after chitooligosaccharides treatments. More importantly, the chitooligosaccharides elicitor strongly stimulated the accumulation of vindoline and catharanthine in the leaves, especially with the treatment of 0.1 µg/mL 3 kDa chitooligosaccharides, the contents of them were increased by 60.68% and 141.54%, respectively. Furthermore, as the defensive responses, antioxidant enzymes activities (catalase, glutathione reductase, ascorbate peroxidase, peroxidase and superoxide dismutase) were enhanced under chitooligosaccharides treatments. To further elucidate the underlying mechanism, qRT-PCR was used to investigate the genes expression levels of secologanin synthase (SLS), strictosidine synthase (STR), strictosidine glucosidase (SGD), tabersonine 16-hydroxylase (T16H), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and octadecanoid-responsive Catharanthus AP2-domain protein 3 (ORCA3). All the genes were significantly up-regulated after chitooligosaccharides treatments, and the transcription abundance of ORCA3, SLS, STR, DAT and PRX1 reached a maximal level with 0.1 µg/mL 3 kDa chitooligosaccharides treatment. All these results suggest that spraying Catharanthus roseus leaves with chitooligosaccharides, especially 0.1 µg/mL of 3 kDa chitooligosaccharides, may effectively improve the pharmaceutical value of Catharanthus roseus.

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.