Knockout of the S-acyltransferase Gene, PbPAT14, Confers the Dwarf Yellowing Phenotype in First Generation Pear by ABA Accumulation.

The development of dwarf fruit trees with smaller and compact characteristics leads to significantly increased fruit production, which is a major objective of pear (Pyrus bretschneideri) breeding. We identified the S-acylation activity of PbPAT14, an S-acyltransferase gene related to plant development, using a yeast (Saccharomyces cerevisiae) complementation assay, and also PbPAT14 could rescue the growth defect of the Arabidopsis mutant atpat14. We further studied the function of PbPAT14 by designing three guide RNAs for PbPAT14 to use in the CRISPR/Cas9 system. We obtained 22 positive transgenic pear lines via Agrobacterium-mediated transformation using cotyledons from seeds of Pyrus betulifolia ('Duli'). Six of these lines exhibited the dwarf yellowing phenotype and were homozygous mutations according to sequencing analysis. Ultrastructure analysis suggested that this dwarfism was manifested by shorter, thinner stems due to a reduction in cell number. A higher level of endogenous abscisic acid (ABA) and a higher transcript level of the ABA pathway genes in the mutant lines revealed that the PbPAT14 function was related to the ABA pathway. Overall, our experimental results increase the understanding of how PATs function in plants and help elucidate the mechanism of plant dwarfism.

A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system.

Pre-planting testing of seeds and plantlets for the existence of quarantine pathogens is an important phytosanitary measure. The CRISPR-mediated molecular diagnostic methodologies are being developed for pathogens detection, but many challenges remain. Here, we profiled an engineered Crispr/LbCas12a variant (LbCas12a-5M) that has more robust trans-cleavage activity and a wider PAM sequences (TNTN) preference than wild type. We developed a procedure for screening specific sequences of bacterial plant pathogens, and the designed species-specific crRNA displayed no cross-reactions with other bacterial species. Combined with a simple extraction of bacterial DNA, an LbCas12a-5M-based visual detection technique was established and optimized for detecting quarantine pathogens Erwinia amylovora and Acidovorax citrulli with detection limits up to 40 CFU/reaction and a sensitivity consistent with qPCR assay. This protocol was faster and simpler than qPCR, requiring 40 min or less from sample preparation. We further validated the potential application of the method by showing that it can be used for rapid and accurate diagnosis of A. citrulli on seeds of watermelon, with 100% agreement with the results of qPCR assay. The developed method simplifies the detection of pathogens and provides cost-effective countermeasures to quarantine interventions.

Genome-Wide Investigation of the Zinc Finger-Homeodomain Family Genes Reveals Potential Roles in Apple Fruit Ripening.

Zinc finger-homeodomain (ZF-HD) transcription factors play an important role in the regulation of plant growth and development, as well as the regulation of stress responses. Studies on the ZF-HD family genes have been conducted in many plants, however, the characteristics of this family in apple (Malus domestica) fruit remains to be poorly understood. In this study, we identified nineteen ZF-HD family genes in apple at the whole-genome scale, which were unevenly located on ten chromosomes. These MdZF-HD genes were phylogenetically divided into two subfamilies: zinc finger-homeodomain (ZHD) and MINI ZINC FINGER (MIF), and the ZHD subfamily was further classified into five groups (ZHDI-ZHDV). Analysis of the gene structures showed that most MdZF-HD genes lack introns. Gene expression analysis indicated that nine selected MdZF-HD genes were differentially responsive to 1-MCP (1-methylcyclopropene) treatment during the postharvest storage of "Qinguan" apple fruit. Moreover, the transcripts of six genes were further validated in "Golden Delicious" apple fruit, and five genes (MdZHD1/2/6/10/11) were significantly repressed and one gene (MdZHD7) was slightly induced by ethylene treatment. These results indicated that these six MdZF-HD genes may involve in the regulation of ethylene induced ripening process of postharvest apple fruit. These findings provide new clues for further functional investigation of ZF-HD genes, such as their roles in the regulation of fruit ripening.

Phenylalanine 4-Hydroxylase Contributes to Endophytic Bacterium Pseudomonas fluorescens' Melatonin Biosynthesis.

Melatonin acts both as an antioxidant and as a growth regulatory substance in plants. Pseudomonas fluorescens endophytic bacterium has been shown to produce melatonin and increase plant resistance to abiotic stressors through increasing endogenous melatonin. However, in bacteria, genes are still not known to be melatonin-related. Here, we reported that the bacterial phenylalanine 4-hydroxylase (PAH) may be involved in the 5-hydroxytryptophan (5-HTP) biosynthesis and further influenced the subsequent production of melatonin in P. fluorescens. The purified PAH protein of P. fluorescens not only hydroxylated phenylalanine but also exhibited l-tryptophan (l-Trp) hydroxylase activity by converting l-Trp to 5-HTP in vitro. However, bacterial PAH displayed lower activity and affinity for l-Trp than l-phenylalanine. Notably, the PAH deletion of P. fluorescens blocked melatonin production by causing a significant decline in 5-HTP levels and thus decreased the resistance to abiotic stress. Overall, this study revealed a possible role for bacterial PAH in controlling 5-HTP and melatonin biosynthesis in bacteria, and expanded the current knowledge of melatonin production in microorganisms.

Genome-wide identification of the PEBP genes in pears and the putative role of PbFT in flower bud differentiation.

Although Phosphatidylethanolamine-binding protein (PEBP) genes have been identified in several plants, little is known about PEBP genes in pears. In this study, a total of 24 PEBP genes were identified, in which 10, 5 and 9 were from Pyrus bretschneideri genome, Pyrus communis genome and Pyrus betuleafolia genome, respectively. Subsequently, gene structure, phylogenetic relationship, chromosomal localization, promoter regions, collinearity and expression were determined with these PEBP genes. It was found that only PbFT from PEBP genes of P. bretschneideri was relatively highly expressed in leaves during flower bud differentiation. Whereas, expression patterns of TFL1 homologues, gene23124 and gene16540, were different from PbFT in buds. The expression pattern and the treatment of reduction day-length indicated that the expression of PbFT in leaves were regulated by day-length and circadian clock. Additionally, the phenotype of transgenic Arabidopsis suggested that PbFT played a role in not only promoting flower bud differentiation, but also regulating the balance between vegetative and reproductive growth. These results may provide important information for further understanding of the evolution and function of PEBP genes in pears.

Investigation of Salt Tolerance Mechanisms Across a Root Developmental Gradient in Almond Rootstocks.

The intensive use of groundwater in agriculture under the current climate conditions leads to acceleration of soil salinization. Given that almond is a salt-sensitive crop, selection of salt-tolerant rootstocks can help maintain productivity under salinity stress. Selection for tolerant rootstocks at an early growth stage can reduce the investment of time and resources. However, salinity-sensitive markers and salinity tolerance mechanisms of almond species to assist this selection process are largely unknown. We established a microscopy-based approach to investigate mechanisms of stress tolerance in and identified cellular, root anatomical, and molecular traits associated with rootstocks exhibiting salt tolerance. We characterized three almond rootstocks: Empyrean-1 (E1), Controller-5 (C5), and Krymsk-86 (K86). Based on cellular and molecular evidence, our results show that E1 has a higher capacity for salt exclusion by a combination of upregulating ion transporter expression and enhanced deposition of suberin and lignin in the root apoplastic barriers, exodermis, and endodermis, in response to salt stress. Expression analyses revealed differential regulation of cation transporters, stress signaling, and biopolymer synthesis genes in the different rootstocks. This foundational study reveals the mechanisms of salinity tolerance in almond rootstocks from cellular and structural perspectives across a root developmental gradient and provides insights for future screens targeting stress response.

PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in Populus.

ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) plays an important role in stress responses, but the transcriptional regulation of ZAT12 in response to abiotic stress remains unclear. In this study, we confirmed that a SALT TOLERANCE ZINC FINGER1 transcription factor from Populus euphratica (PeSTZ1) could regulate the expression of PeZAT12 by dual-luciferase reporter (DLR) assay and electrophoretic mobility shift assay. The expression of PeSTZ1 was rapidly induced by NaCl and hydrogen peroxide (H2O2) treatments. Overexpressing PeSTZ1 in poplar 84K (Populus alba × Populus glandulosa) plant was endowed with a strong tolerance to salt stress. Under salt stress, transgenic poplar exhibited higher expression levels of PeZAT12 and accumulated a larger amount of antioxidant than the wild-type plants. Meanwhile, ASCORBATE PEROXIDASE2 (PeAPX2) can be activated by PeZAT12 and PeSTZ1, promoting the accumulation of cytosolic ascorbate peroxidase (APX) to scavenge reactive oxygen species (ROS) under salt stress. This new regulatory model (PeSTZ1-PeZAT12-PeAPX2) was found in poplar, providing a new idea and insight for the interpretation of poplar resistance. Transgenic poplar reduced the accumulation of ROS, restrained the degradation of chlorophyll and guaranteed the photosynthesis and electron transport system. On the other hand, transgenic poplar slickly adjusted K+/Na+ homeostasis to alleviate salt toxicity in photosynthetic organs of plants under salt stress and then increased biomass accumulation. In summary, PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in poplar.