An LcMYB111-LcHY5 Module Differentially Activates an LcFLS Promoter in Different Litchi Cultivars.

Flavonol synthase (FLS) is the crucial enzyme of the flavonol biosynthetic pathways, and its expression is tightly regulated in plants. In our previous study, two alleles of LcFLS,LcFLS-A and LcFLS-B, have been identified in litchi, with extremely early-maturing (EEM) cultivars only harboring LcFLS-A, while middle-to-late-maturing (MLM) cultivars only harbor LcFLS-B. Here, we overexpressed both LcFLS alleles in tobacco, and transgenic tobacco produced lighter-pink flowers and showed increased flavonol levels while it decreased anthocyanin levels compared to WT. Two allelic promoters of LcFLS were identified, with EEM cultivars only harboring proLcFLS-A, while MLM cultivars only harbor proLcFLS-B. One positive and three negative R2R3-MYB transcription regulators of LcFLS expression were identified, among which only positive regulator LcMYB111 showed a consistent expression pattern with LcFLS, which both have higher expression in EEM than that of MLM cultivars. LcMYB111 were further confirmed to specifically activate proLcFLS-A with MYB-binding element (MBE) while being unable to activate proLcFLS-B with mutated MBE (MBEm). LcHY5 were also identified and can interact with LcMYB111 to promote LcFLS expression. Our study elucidates the function of LcFLS and its differential regulation in different litchi cultivars for the first time.

A plant-unique ESCRT component, FYVE4, regulates multivesicular endosome biogenesis and plant growth.

During evolution, land plants generated unique proteins that participate in endosomal sorting and multivesicular endosome (MVE) biogenesis, many of them with specific phosphoinositide-binding capabilities. Nonetheless, the function of most plant phosphoinositide-binding proteins in endosomal trafficking remains elusive. Here, we analysed several Arabidopsis mutants lacking predicted phosphoinositide-binding proteins and first identified fyve4-1 as a mutant with a hypersensitive response to high-boron conditions and defects in degradative vacuolar sorting of membrane proteins such as the borate exporter BOR1-GFP. FYVE4 encodes a plant-unique, FYVE domain-containing protein that interacts with SNF7, a core component of ESCRT-III (Endosomal Sorting Complex Required for Transport III). FYVE4 affects the membrane association of the late-acting ESCRT components SNF7 and VPS4, and modulates the formation of intraluminal vesicles (ILVs) inside MVEs. The critical function of FYVE4 in the ESCRT pathway was further demonstrated by the strong genetic interactions with SNF7B and LIP5. Although the fyve4-1, snf7b and lip5 single mutants were viable, the fyve4-1 snf7b and fyve4-1 lip5 double mutants were seedling lethal, with strong defects in MVE biogenesis and vacuolar sorting of ubiquitinated membrane proteins. Taken together, we identified FYVE4 as a novel plant endosomal regulator, which functions in ESCRTing pathway to regulate MVE biogenesis.

Autophagy Mediates the Degradation of Plant ESCRT Component FREE1 in Response to Iron Deficiency.

Multivesicular body (MVB)-mediated endosomal sorting and macroautophagy are the main pathways mediating the transport of cellular components to the vacuole and are essential for maintaining cellular homeostasis. The interplay of these two pathways remains poorly understood in plants. In this study, we show that FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), which was previously identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT), essential for MVB biogenesis and plant growth, can be transported to the vacuole for degradation in response to iron deficiency. The vacuolar transport of ubiquitinated FREE1 protein is mediated by the autophagy pathway. As a consequence, the autophagy deficient mutants, atg5-1 and atg7-2, accumulate more endogenous FREE1 protein and display hypersensitivity to iron deficiency. Furthermore, under iron-deficient growth condition autophagy related genes are upregulated to promote the autophagic degradation of FREE1, thereby possibly relieving the repressive effect of FREE1 on iron absorption. Collectively, our findings demonstrate a unique regulatory mode of protein turnover of the ESCRT machinery through the autophagy pathway to respond to iron deficiency in plants.

SINAT E3 ligases regulate the stability of the ESCRT component FREE1 in response to iron deficiency in plants.

The endosomal sorting complex required for transport (ESCRT) machinery is an ancient, evolutionarily conserved membrane remodeling complex that is essential for multivesicular body (MVB) biogenesis in eukaryotes. FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), which was previously identified as a plant-specific ESCRT component, modulates MVB-mediated endosomal sorting and autophagic degradation. Although the basic cellular functions of FREE1 as an ESCRT component have been described, the regulators that control FREE1 turnover remain unknown. Here, we analyzed how FREE1 homeostasis is mediated by the RING-finger E3 ubiquitin ligases, SINA of Arabidopsis thaliana (SINATs), in response to iron deficiency. Under iron-deficient growth conditions, SINAT1-4 were induced and ubiquitinated FREE1, thereby promoting its degradation and relieving the repressive effect of FREE1 on iron absorption. By contrast, SINAT5, another SINAT member that lacks ubiquitin ligase activity due to the absence of the RING domain, functions as a protector protein which stabilizes FREE1. Collectively, our findings uncover a hitherto unknown mechanism of homeostatic regulation of FREE1, and demonstrate a unique regulatory SINAT-FREE1 module that subtly regulates plant response to iron deficiency stress.

Genome-Wide Development of Polymorphic SNP Markers and Evaluation of Genetic Diversity of Litchi (Litchi chinensis Sonn.).

Litchi (Litchi chinensis Sonn.) is a highly valuable fruit crop that is widely grown in tropical and subtropical areas of the world. Studying its genetic diversity and population structure is critical for effective conservation and breeding programs. In this study, we developed 150 single-nucleotide polymorphism (SNP) markers that were evenly spaced across litchi genome and applied them to the evaluation of the genetic diversity of 84 litchi accessions, including old cultivars, modern cultivars, hybrids from known parents and wild accessions. Ninety-one SNP markers, showing high levels of polymorphism and high genotyping success rates, were used for further analysis. The newly developed SNP markers captured a relatively higher level of genetic diversity (He = 0.364) in litchi cultivars and could be successfully applied for the identification of synonymous cultivars and hybrids with close genetic backgrounds. Cluster analysis grouped all genotypes into three clusters that showed perfect association with their fruit maturation period, among which wild accessions clustered with their corresponding domesticated cultivars, and hybrids from different parent combinations showed different inheritance tendencies. Our study not only provided a set of efficient SNP markers for future genetic research, but also laid an important foundation for the conservation and genetic breeding of litchi.

[Characteristics of Water-soluble Ions in an Autumn Haze Process in the Southern Sichuan Urban Agglomeration After the Implementation of China's Air Pollution Prevention and Control Action Plan].

To investigate the PM2.5 pollution in the southern Sichuan urban agglomeration after the implementation of China's Air Pollution Prevention and Control Action Plan (APPCAP), PM2.5 samples were simultaneously collected in four cities (Neijiang, Zigong, Yibin, and Luzhou) from November 7 to 19, 2018. The pollution characteristics of PM2.5 and main water-soluble ions were analyzed in combination with the synoptic situation, and the influence of regional transport on atmospheric pollution was also discussed in this study. The results showed that the mean ρ(PM2.5) in this region was (67.2±38.3) µg·m-3, being highest in Luzhou and lowest in Neijiang. The proportion of SNA (SO42-, NO3-, and NH4+) in PM2.5 was 33.3%, among which NO3- was dominant. From the intermediate stage (2015) to the end(2018) of the implementation of APPCAP, ρ(PM2.5) values were increased by 13.8%, 47.2%, and 69.1% in Neijiang, Yibin, and Luzhou, respectively, though unchanged in Zigong. Due to the significant reduction in ρ(SO2) but slight decrease or increase in ρ(NO2), as well as the lack of controlling NH3 emissions, from 2015 to 2018, ρ(NO3-) had increased by 36.7%-116.0%, whereas ρ(SO42-) decreased by 19.8%-40.2%, and ρ(NH4+) changed slightly in four cities. On haze days, the nitrogen oxidation rate (NOR) increased by 60.0%-118.2%, whereas the sulfur oxidation rate (SOR) increased slightly or decreased, leading to a significant increase in ρ(NO3-) (2.7-3.0 times that on clean days) and NO3-/SO42- mass ratios (1.7-1.9 on haze days). These values indicated that the secondary formation of nitrate was the dominant chemical mechanism in this haze process. On haze days, the PM2.5 pollution in this region was mainly affected by the regional transport within Sichuan Basin, particularly by the northeasterly air masses passing through Chongqing.

A Combinatorial Reporter Set to Visualize the Membrane Contact Sites Between Endoplasmic Reticulum and Other Organelles in Plant Cell.

The membrane contact sites (MCSs) enable interorganelle communication by associating organelles at distances of tens of nanometers over extended membrane surfaces and serve to maintain cellular homeostasis through efficient exchange of metabolites, lipid, and calcium between organelles, organelle fission, and movement. Most MCSs and a growing number of tethering proteins especially those involved in mediating the junctions between endoplasmic reticulum (ER) and other organelles have been extensively characterized in mammal and yeast. However, the studies of plant MCSs are still at stages of infancy, at least one reason might be due to the lack of bona fide markers for visualizing these membrane junctions in plant cells. In this study, a series of genetically encoded reporters using split super-folder GFP protein were designed to detect the possible MCSs between ER and three other cellular compartments including chloroplast, mitochondria and plasma membrane (PM) in plant cell. By expressing these genetically encoded reporter in Arabidopsis protoplasts as well as Nicotiana benthamiana leaf, we could intuitively observe the punctate signal surrounding chloroplast upon expression of ER-chloroplast MCS reporter, punctate signal of ER-mitochondria MCS reporter and punctate signal close to the PM upon expression of ER-PM MCS reporter. We also showed that the ER-chloroplast MCSs were dynamic structures that undergo active remodeling with concomitant occurrence of chloroplast dysfunction inside plant cells. This study demonstrates that ER associates with various organelles in close proximity in plant cells and provides tools that might be applicable for visualizing MCSs in plants.

Cloning and Characterization of a Flavonol Synthase Gene From Litchi chinensis and Its Variation Among Litchi Cultivars With Different Fruit Maturation Periods.

Litchi (Litchi chinensis) is an important subtropical fruit tree with high commercial value. However, the short and centralized fruit maturation period of litchi cultivars represents a bottleneck for litchi production. Therefore, the development of novel cultivars with extremely early fruit maturation period is critical. Previously, we showed that the genotypes of extremely early-maturing (EEM), early-maturing (EM), and middle-to-late-maturing (MLM) cultivars at a specific locus SNP51 (substitution type C/T) were consistent with their respective genetic background at the whole-genome level; a homozygous C/C genotype at SNP51 systematically differentiated EEM cultivars from others. The litchi gene on which SNP51 was located was annotated as flavonol synthase (FLS), which catalyzes the formation of flavonols. Here, we further elucidate the variation of the FLS gene from L. chinensis (LcFLS) among EEM, EM, and MLM cultivars. EEM cultivars with a homozygous C/C genotype at SNP51 all contained the same 2,199-bp sequence of the LcFLS gene. For MLM cultivars with a homozygous T/T genotype at SNP51, the sequence lengths of the LcFLS gene were 2,202-2,222 bp. EM cultivars with heterozygous C/T genotypes at SNP51 contained two different alleles of the LcFLS gene: a 2,199-bp sequence identical to that in EEM cultivars and a 2,205-bp sequence identical to that in MLM cultivar 'Heiye.' Moreover, the coding regions of LcFLS genes of other MLM cultivars were almost identical to that of 'Heiye.' Therefore, the LcFLS gene coding region may be used as a source of diagnostic SNP markers to discriminate or identify genotypes with the EEM trait. The expression pattern of the LcFLS gene and accumulation pattern of flavonol from EEM, EM, and MLM cultivars were analyzed and compared using quantitative real-time PCR (qRT-PCR) and high-performance liquid chromatography (HPLC) for mature leaves, flower buds, and fruits, 15, 30, 45, and 60 days after anthesis. Flavonol content and LcFLS gene expression levels were positively correlated in all three cultivars: both decreased from the EEM to MLM cultivars, with moderate levels in the EM cultivars. LcFLS gene function could be further analyzed to elucidate its correlation with phenotype variation among litchi cultivars with different fruit maturation periods.

Analysis of Golgi-Mediated Protein Traffic in Plant Cells.

In plant secretory pathways, the Golgi apparatus serves as the major sorting hub to receive de novo synthesized protein from the endoplasmic reticulum for further sorting to post-Golgi compartments or for residence in the cisternae of Golgi stacks. Meanwhile, Golgi functions as a pivotal biochemical factory to make modifications of N-glycans and to produce mature glycoproteins. Fluorescent tag-based confocal microscopy in combination with the brefeldin A drug or the genetic tools to disturb Golgi function have been shown as powerful approaches to analyze Golgi-mediated protein traffic in transiently expressed plant protoplasts or in stably expressed transgenic plants. Various endoglycosidases like Endo H and PNGase F have been widely used to monitor Golgi-mediated glycosylation of secretory proteins. Here, using fluorescently tagged Golgi-resident proteins and known glycosylated proteins as examples, we describe detailed protocols to analyze Golgi-mediated protein traffic and glycosylation in transiently expressed protoplasts derived from Arabidopsis suspension culture cells and in stably expressed transgenic plants.

Identifying Litchi (Litchi chinensis Sonn.) Cultivars and Their Genetic Relationships Using Single Nucleotide Polymorphism (SNP) Markers.

Litchi is an important fruit tree in tropical and subtropical areas of the world. However, there is widespread confusion regarding litchi cultivar nomenclature and detailed information of genetic relationships among litchi germplasm is unclear. In the present study, the potential of single nucleotide polymorphism (SNP) for the identification of 96 representative litchi accessions and their genetic relationships in China was evaluated using 155 SNPs that were evenly spaced across litchi genome. Ninety SNPs with minor allele frequencies above 0.05 and a good genotyping success rate were used for further analysis. A relatively high level of genetic variation was observed among litchi accessions, as quantified by the expected heterozygosity (He = 0.305). The SNP based multilocus matching identified two synonymous groups, 'Heiye' and 'Wuye', and 'Chengtuo' and 'Baitangli 1'. A subset of 14 SNPs was sufficient to distinguish all the non-redundant litchi genotypes, and these SNPs were proven to be highly stable by repeated analyses of a selected group of cultivars. Unweighted pair-group method of arithmetic averages (UPGMA) cluster analysis divided the litchi accessions analyzed into four main groups, which corresponded to the traits of extremely early-maturing, early-maturing, middle-maturing, and late-maturing, indicating that the fruit maturation period should be considered as the primary criterion for litchi taxonomy. Two subpopulations were detected among litchi accessions by STRUCTURE analysis, and accessions with extremely early- and late-maturing traits showed membership coefficients above 0.99 for Cluster 1 and Cluster 2, respectively. Accessions with early- and middle-maturing traits were identified as admixture forms with varying levels of membership shared between the two clusters, indicating their hybrid origin during litchi domestication. The results of this study will benefit litchi germplasm conservation programs and facilitate maximum genetic gains in litchi breeding programs.