High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa).

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature

The effects of preharvest LED light, melatonin and AVG treatments on the quality of postharvest snapdragon and vase life

Snapdragon (Antirrhinum majus) is one of top ten fresh-cut flowers in the United States; however, its short vase life limits its marketability. The purposes of this study were to test the effects of LED light, exogenous melatonin and one ethylene production inhibitor, AVG, on the quality of pre-harvest snapdragon and the prolongation of vase life after post-harvesting. Our results showed that snapdragon treated with 10 h white light followed by 6 h blue light (WB) inhibited stem elongation and lengths of the inflorescences, reduced the number of florets and vase life. On the contrary, snapdragon treated with 10 h white light, 3 h red light, 3 h blue light (WRB) significantly promoted stem elongation, lengths of the inflorescences, and increased the size and number of florets. The lengths of stems and inflorescences increased significantly in all melatonin treatments while quantity and size of florets only increased with 200 µmol·L-1 melatonin application. Noticeably, vase life was significantly extended with 200 µmol·L-1 melatonin application and shortened with WB treatment. In contrast to melatonin, all AVG treatments resulted in decreases of the floret size; and changes in stem elongation and inflorescence length were only observed in the treatment with 100 µmol·L-1AVG. These results showed that pre-harvest treatment with WBR and melatonin can effectively improve the post-harvest quality of snapdragon flowers and 200 µmol·L-1 of melatonin extended their vase life

Physiological and Transcriptome Analyses Reveal Short-Term Responses and Formation of Memory Under Drought Stress in Rice

In some plants, exposure to stress can induce a memory response, which appears to play an important role in adaptation to recurrent stress environments. However, whether rice exhibits drought stress memory and the molecular mechanisms that might underlie this process have remained unclear. Here, we ensured that rice drought memory was established after cycles of mild drought and re-watering treatment, and studied gene expression by whole-transcriptome strand-specific RNA sequencing (ssRNA-seq). We detected 6,885 transcripts and 238 lncRNAs involved in the drought memory response, grouped into 16 distinct patterns. Notably, the identified genes of dosage memory generally did not respond to the initial drought treatment. Our results demonstrate that stress memory can be developed in rice under appropriate water deficient stress, and lncRNA, DNA methylation and endogenous phytohormones (especially abscisic acid) participate in rice short-term drought memory, possibly acting as memory factors to activate drought-related memory transcripts in pathways such as photosynthesis and proline biosynthesis, to respond to the subsequent stresses

Antioxidant response and related gene expression in aged oat seed

To evaluate deterioration of oat seeds during storage, we analyzed oxygen radicals, antioxidant enzyme activity, proline content, and gene transcript levels in oat seeds with different moisture contents (4%, 16%, and 28% w/w) during storage for 0, 6, and 12 months (CK, LT-6, and LT-12 treatments, respectively) at 4 °C. The germination percentage decreased significantly with higher seed moisture contents and longer storage duration. The concentrations of superoxide radical and hydrogen peroxide increased with seed moisture content increasing. The activities of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) may have had a complementary or interacting role to scavenge reactive oxygen species. As the storage duration extended, the proline content decreased in seeds with 4% and 16% moisture content and increased in 28%. These findings suggest that proline played the main role in adaptation to oxidative stress in seeds with higher moisture content (28%), while antioxidant enzymes played the main role in seeds with lower moisture contents (4%, 16%). In the gene transcript analyses, SOD1 transcript levels were not consistent with total SOD activity. The transcript levels of APX1 and CAT1 showed similar trends to those of APX and CAT activity. The transcript levels of P5CS1, which encodes a proline biosynthetic enzyme, increased with seed moisture content increasing in CK. Compared with changing of proline content in seeds stored 12 months, PDH1 transcript levels showed the opposite trend and maintained the lower levels in seeds of 16% and 28% moisture contents. The transcript level of P5CS1 was significantly affected by moisture content, and PDH1 could improve stress resistance for seed aging and maintain seed vigor during long-term storage

Effect of Nanocrystallization of Anthocyanins Extracted from Two Types of Red-Fleshed Apple Varieties on Its Stability and Antioxidant Activity

Red-fleshed apple (Malus sieversii f. neidzwetzkyana (Dieck) Langenf) has attracted more and more attention due to its enriched anthocyanins and high antioxidant activity. In this study we extracted total anthocyanins and phenols from two types of red-fleshed apples—Xinjing No.4 (XJ4) and Red Laiyang (RL)—to study the stability and antioxidant activity of anthocyanins after encapsulation onto Corn Starch Nanoparticles (CSNPs). The results indicated the anthocyanins and total phenol levels of XJ4 were 2.96 and 2.25 times higher than those of RL respectively. The anthocyanin concentration and loading time had a significant effect on CSNPs encapsulation, and XJ4 anthocyanins always showed significantly higher loading capacity than RL. After encapsulation, the morphology of RL-CSNPs and XJ4-CSNPs was still spherical with a smooth surface as CSNPs, but the particle size increased compared to CSNPs especially for RL-CSNPs. Different stress treatments including UV light, pH, temperature, and salinity suggested that XJ4-CSNPs exhibited consistently higher stability than RL-CSNPs. A significantly enhanced free radical scavenging rate under stress conditions was observed, and XJ4-CSNPs had stronger antioxidant activity than RL-CSNPs. Furthermore, XJ4-CSNPs exhibited a slower released rate than RL-CSNPs in simulated gastric (pH 2.0) and intestinal (pH 7.0) environments. Our research suggests that nanocrystallization of anthocyanins is an effective method to keep the anthocyanin ingredients intact and active while maintaining a slow release rate. Compared to RL, encapsulation of XJ4 anthocyanins has more advantages, which might be caused by the significant differences in the metabolites of XJ4. These findings give an insight into understanding the role of nanocrystallization using CSNPs in enhancing the antioxidant ability of anthocyanins from different types of red-fleshed apples, and provide theoretical foundations for red-fleshed apple anthocyanin application

Photosynthetic Responses of Anthurium × ‘Red’ under Different Light Conditions

Light is an essential energy source for plant photosynthesis, although it can also be a stress-causing element. Therefore, the current research was aimed to compare photosynthetic responses of Anthurium × ‘Red’ leaves at different positions (bottom old leaf, 1; center mature leaf, 2; top expanded leaf, 3) established under three photosynthetic photon flux densities (PPFDs): 550 μmol·m−2·s−1 as high (H), 350 μmol·m−2·s−1 as medium (M), and 255 μmol·m−2·s−1 as low (L). After six months, all the replicates were relocated to interior rooms with a PPFD of 30 μmol·m−2·s−1. There were no significant differences in chlorophyll concentration of the old leaf among treatments, before (Day 0) and after shifting the plants to interior rooms (Day 30). The total chlorophyll concentrations of the mature and top leaves increased significantly. In greenhouse conditions, H and M treatments did not show any significant change for net photosynthetic rate (Pn) at various leaf positions. However, M2 exhibited an improved Pn in the interior conditions. Plants grown under M treatment were greener and had bigger leaves compared to other treatments. Our study reveals that Anthurium × ‘Red’ photosynthesis responses to different light conditions varied distinctly. However, M treatment can keep the plants looking green by accumulating enough energy for indoor conditions, and middle and lower leaves may be triggered to restore photosynthetic activity under low light or indoor conditions

Nanocrystallization of Anthocyanin Extract from Red-Fleshed Apple ′QN-5′ Improved Its Antioxidant Effect through Enhanced Stability and Activity under Stressful Conditions

Red-flesh apples are known as functional fruits because of their rich anthocyanin. The anthocyanin content of the red flesh apple cultivar ′QN-5′ we bred can reach 361 mg·kg−1 (FW), and showed higher scavenging capacity to DPPH radicals, hydroxyl radicals, and superoxide anion radicals, with scavenging rates of 80.0%, 54.0%, and 43.3%, respectively. We used this particular anthocyanin-rich ′QN-5′ apple as material to examine how nanocrystallization affects the antixodiant effect of anthocyanin. The anthocyanin extract was encapsulated with biocompatible zein to form zein-anthocyanin nanoparticles (ZANPs). Transmission electron microscopy (TEM) scanning showed that ZANPs had a regular spherical shape with an average diameter size of 50–60nm. When the ratio of the zein and the anthocyanin was 1:0.5, the results suggested that the encapsulation efficiency (EE) of the ZANPs could reach as high as 92.8%, and that scavenging rate for DPPH radicals was increased from 87.1% to 97.2% compared to the non-nanocrystallized anthocyanin extract. Interestingly, treatment under alkaline conditions (pH 9.0), high temperature (90 °C), and a storage time of 7 days could decrease the scavenging capacity of the ZANPs for DPPH radicals, but this scavenging capacity loss for ZANPs was significantly lower than that observed in the non-nanocrystallized anthocyanin, suggesting the higher stability of ZANPs is caused by encapsulation. These results would provide a theoretical basis for the application of the anthocyanin in scavenging free radicals under stress conditions