Front Cover Image,Volume 116, Number 11, November 2019

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome ( www.ibiodesign.net/gBIG ).     

Alleviation of the detrimental effect of water deficit on wheat (Triticum aestivum L.) growth by an indole acetic acid-producing endophytic fungus

Plant and Soil - Endophytic fungi colonization is an eco-friendly strategy to respond to environmental stresses and confer tolerance to the host plant. Here, the responses of wheat plant inoculated...     

Genome‐wide association study identifies variation of glucosidase being linked to natural variation of the maximal quantum yield of photosystem II

The maximum quantum yield of photosystem II (as reflected by variable to maximum chlorophyll a fluorescence, F_v/F_m) is regarded as one of the most important photosynthetic parameters. The genetic basis underlying natural variation in F_v/F_m, which shows low level of variations in plants under non‐stress conditions, is not easy to be exploited using the conventional gene cloning approaches. Thus, in order to answer this question, we have followed another strategy: we used genome‐wide association study (GWAS) and transgenic analysis in a rice mini‐core collection. We report here that four single‐nucleotide polymorphisms, located in the promoter region of β‐glucosidase 5 (BGlu‐5), are associated with observed variation in F_v/F_m. Indeed, our transgenic analysis showed a good correlation between BGlu‐5 and F_v/F_m. Thus, our work demonstrates the feasibility of using GWAS to study natural variation in F_v/F_m, suggesting that cis‐element polymorphism, affecting the BGlu‐5 expression level, may, indirectly, contribute to F_v/F_m variation in rice through the gibberellin signaling pathway. Further research is needed to understand the mechanism of our novel observation.     

Physiological,Biochemical and Molecular Responses of Barley Seedlings to Aluminum Stress

Barley (Hordeum vulgare L.) is one of the most Aluminum (Al) sensitive cereal species. In this study, the physiological, biochemical, and molecular response of barley seedlings to Al treatment was examined to gain insight into Al response and tolerance mechanisms. The results showed that superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were inhibited to different degrees following Al exposure. The MDA content also significantly increased with increasing Al concentrations. SRAP results indicated significant differences between Al treatments and controls in terms of SRAP profile, and the genomic template stability (GTS) decreased with increasing Al concentration and duration. These integrative results help to elucidate the underlying mechanisms that the barley response to Al toxicity.     

Golgi‐localized cation/proton exchangers regulate ionic homeostasis and skotomorphogenesis in Arabidopsis

Multiple transporters and channels mediate cation transport across the plasma membrane and tonoplast to regulate ionic homeostasis in plant cells. However, much less is known about the molecular function of transporters that facilitate cation transport in other organelles such as Golgi. We report here that Arabidopsis KEA4, KEA5, and KEA6, members of cation/proton antiporters‐2 (CPA2) superfamily were colocalized with the known Golgi marker, SYP32‐mCherry. Although single kea4,5,6 mutants showed similar phenotype as the wild type under various conditions, kea4/5/6 triple mutants showed hypersensitivity to low pH, high K⁺, and high Na⁺ and displayed growth defects in darkness, suggesting that these three KEA‐type transporters function redundantly in controlling etiolated seedling growth and ion homeostasis. Detailed analysis indicated that the kea4/5/6 triple mutant exhibited cell wall biosynthesis defect during the rapid etiolated seedling growth and under high K⁺/Na⁺ condition. The cell wall‐derived pectin homogalacturonan (GalA)₃ partially suppressed the growth defects and ionic toxicity in the kea4/5/6 triple mutants when grown in the dark but not in the light conditions. Together, these data support the hypothesis that the Golgi‐localized KEAs play key roles in the maintenance of ionic and pH homeostasis, thereby facilitating Golgi function in cell wall biosynthesis during rapid etiolated seedling growth and in coping with high K⁺/Na⁺ stress.     

Analysis of catalase mutants underscores the essential role of CATALASE2 for plant growth and day length‐dependent oxidative signalling

Three genes encode catalase in Arabidopsis. Although the role of CAT2 in photorespiration is well established, the importance of the different catalases in other processes is less clear. Analysis of cat1, cat2, cat3, cat1 cat2, and cat2 cat3 T‐DNA mutants revealed that cat2 had the largest effect on activity in both roots and leaves. Root growth was inhibited in all cat2‐containing lines, but this inhibition was prevented by growing plants at high CO₂, suggesting that it is mainly an indirect effect of stress at the leaf level. Analysis of double mutants suggested some overlap between CAT2 and CAT3 functions in leaves and CAT1 and CAT2 in seeds. When plants had been grown to a similar developmental stage in short days or long days, equal‐time exposure to oxidative stress caused by genetic or pharmacological inhibition of catalase produced a much stronger induction of H₂O₂ marker genes in short day plants. Together, our data (a) underline the importance of CAT2 in basal H₂O₂ processing in Arabidopsis; (b) suggest that CAT1 and CAT3 are mainly “backup” or stress‐specific enzymes; and (c) establish that day length‐dependent responses to catalase deficiency are independent of the duration of oxidative stress.     

Vacuolar membrane structures and their roles in plant–pathogen interactions

Plant Molecular Biology - Short review focussing on the role and targeting of vacuolar substructure in plant immunity and pathogenesis. Plants lack specialized immune cells, therefore each plant...