[Repair of distal phalanx finger wound with modified great toe fibular flap with distal artery pedicle as reflux vein].

OBJECTIVE: To investigate the effectiveness of the modified great toe fibular flap using the distal artery pedicle as reflux vein for repairing distal phalanx finger wound. METHODS: Between June 2018 and January 2020, 15 patients who suffered tissue defect of the distal phalanx finger were treated, including 12 males and 3 females, the average age was 40.2 years (range, 24-56 years). All of them were caused by machine crush injury. There were 2 cases of thumb, 6 cases of index finger, 3 cases of middle finger, 3 cases of ring finger, and 1 case of little finger. The defects ranged from 1.7 cm×1.3 cm to 3.0 cm×2.0 cm. The time from injury to admission was 0.6-4 hours, with an average of 2.3 hours. The medial fibular proper digital artery was further dissociated to the distal end and anastomosed with the recipient vein as the reflux vein. The area of flaps ranged from 2.0 cm×1.5 cm to 3.2 cm×2.2 cm. RESULTS: All the flaps survived without vascular crisis, and the wounds healed by first intention. Except for 1 case that the suture was too tight, the incision was partially split after the stitches were removed, and it healed spontaneously after dressing change, the other patients had good healing of the donor site incision and normal foot function. All 15 patients were followed up 3-18 months, with an average of 9.3 months. The appearance of finger pulps were satisfactory with full and threaded. The color, texture, and elasticity of the flaps were good, and the two-point discrimination was 6-8 mm at last follow-up. The flexion and extension of fingers were normal. At last follow-up, hand function was evaluated according to the upper limb function evaluation trial standard of the Chinese Medical Association Hand Surgery Society, and the results were 13 cases of excellent and 2 cases of good. CONCLUSION: Modified great toe fibular flap using the distal arterial pedicle as a reflux vein can improve the venous drainage of the flap and contribute to increase the success rate of the flap without additional injury.

Lipophilic signals lead to organ-specific gene expression changes in Arabidopsis seedlings.

In plants, N-acylethanolamines (NAEs) are most abundant in desiccated seeds and their levels decline during germination and early seedling establishment. However, endogenous NAE levels rise in seedlings when ABA or environmental stress is applied, and this results in an inhibition of further seedling development. When the most abundant, polyunsaturated NAEs of linoleic acid (18:2) and linolenic acid (18:3) were exogenously applied, seedling development was affected in an organ-specific manner. NAE 18:2 primarily affected primary root elongation and NAE 18:3 primarily affected cotyledon greening and expansion and overall seedling growth. The molecular components and signaling mechanisms involved in this pathway are not well understood. In addition, the bifurcating nature of this pathway provides a unique system in which to study the spatial aspects and interaction of these lipid-specific and organ-targeted signaling pathways. Using whole transcriptome sequencing (RNA-seq) and differential expression analysis, we identified early (1-3 hr) transcriptional changes induced by the exogenous treatment of NAE 18:2 and NAE 18:3 in cotyledons, roots, and seedlings. These two treatments led to a significant enrichment in ABA-response and chitin-response genes in organs where the treatments led to changes in development. In Arabidopsis seedlings, NAE 18:2 treatment led to the repression of genes involved in cell wall biogenesis and organization in roots and seedlings. In addition, cotyledons, roots, and seedlings treated with NAE 18:3 also showed a decrease in transcripts that encode proteins involved in growth processes. NAE 18:3 also led to changes in the abundance of transcripts involved in the modulation of chlorophyll biosynthesis and catabolism in cotyledons. Overall, NAE 18:2 and NAE 18:3 treatment led to lipid-type and organ-specific gene expression changes that include overlapping and non-overlapping gene sets. These data will provide future, rich opportunities to examine the genetic pathways involved in transducing early signals into downstream physiological changes in seedling growth.

Seedling Chloroplast Responses Induced by N-Linolenoylethanolamine Require Intact G-Protein Complexes.

In animals, several long-chain N-acylethanolamines (NAEs) have been identified as endocannabinoids and are autocrine signals that operate through cell surface G-protein-coupled cannabinoid receptors. Despite the occurrence of NAEs in land plants, including nonvascular plants, their precise signaling properties and molecular targets are not well defined. Here we show that the activity of N-linolenoylethanolamine (NAE 18:3) requires an intact G-protein complex. Specifically, genetic ablation of the Gßγ dimer or loss of the full set of atypical Gα subunits strongly attenuates an NAE-18:3-induced degreening of cotyledons in Arabidopsis (Arabidopsis thaliana) seedlings. This effect involves, at least in part, transcriptional regulation of chlorophyll biosynthesis and catabolism genes. In addition, there is feedforward transcriptional control of G-protein signaling components and G-protein interactors. These results are consistent with NAE 18:3 being a lipid signaling molecule in plants with a requirement for G-proteins to mediate signal transduction, a situation similar, but not identical, to the action of NAE endocannabinoids in animal systems.

Starch Deficiency Enhances Lipid Biosynthesis and Turnover in Leaves.

Starch and lipids represent two major forms of carbon and energy storage in plants and play central roles in diverse cellular processes. However, whether and how starch and lipid metabolic pathways interact to regulate metabolism and growth are poorly understood. Here, we show that lipids can partially compensate for the lack of function of transient starch during normal growth and development in Arabidopsis (Arabidopsis thaliana). Disruption of starch synthesis resulted in a significant increase in fatty acid synthesis via posttranslational regulation of the plastidic acetyl-coenzyme A carboxylase and a concurrent increase in the synthesis and turnover of membrane lipids and triacylglycerol. Genetic analysis showed that blocking fatty acid peroxisomal ß-oxidation, the sole pathway for metabolic breakdown of fatty acids in plants, significantly compromised or stunted the growth and development of mutants defective in starch synthesis under long days or short days, respectively. We also found that the combined disruption of starch synthesis and fatty acid turnover resulted in increased accumulation of membrane lipids, triacylglycerol, and soluble sugars and altered fatty acid flux between the two lipid biosynthetic pathways compartmentalized in either the chloroplast or the endoplasmic reticulum. Collectively, our findings provide insight into the role of fatty acid ß-oxidation and the regulatory network controlling fatty acid synthesis, and they reveal the mechanistic basis by which starch and lipid metabolic pathways interact and undergo cross talk to modulate carbon allocation, energy homeostasis, and plant growth.

Arabidopsis TRIGALACTOSYLDIACYLGLYCEROL5 Interacts with TGD1, TGD2, and TGD4 to Facilitate Lipid Transfer from the Endoplasmic Reticulum to Plastids.

The biogenesis of photosynthetic membranes in the plastids of higher plants requires an extensive supply of lipid precursors from the endoplasmic reticulum (ER). Four TRIGALACTOSYLDIACYLGLYCEROL (TGD) proteins (TGD1,2,3,4) have thus far been implicated in this lipid transfer process. While TGD1, TGD2, and TGD3 constitute an ATP binding cassette transporter complex residing in the plastid inner envelope, TGD4 is a transmembrane lipid transfer protein present in the outer envelope. These observations raise questions regarding how lipids transit across the aqueous intermembrane space. Here, we describe the isolation and characterization of a novel Arabidopsis thaliana gene, TGD5. Disruption of TGD5 results in similar phenotypic effects as previously described in tgd1,2,3,4 mutants, including deficiency of ER-derived thylakoid lipids, accumulation of oligogalactolipids, and triacylglycerol. Genetic analysis indicates that TGD4 is epistatic to TGD5 in ER-to-plastid lipid trafficking, whereas double mutants of a null tgd5 allele with tgd1-1 or tgd2-1 show a synergistic embryo-lethal phenotype. TGD5 encodes a small glycine-rich protein that is localized in the envelope membranes of chloroplasts. Coimmunoprecipitation assays show that TGD5 physically interacts with TGD1, TGD2, TGD3, and TGD4. Collectively, these results suggest that TGD5 facilitates lipid transfer from the outer to the inner plastid envelope by bridging TGD4 with the TGD1,2,3 transporter complex.

Arabidopsis lipins, PDAT1 acyltransferase, and SDP1 triacylglycerol lipase synergistically direct fatty acids toward ß-oxidation, thereby maintaining membrane lipid homeostasis.

Triacylglycerol (TAG) metabolism is a key aspect of intracellular lipid homeostasis in yeast and mammals, but its role in vegetative tissues of plants remains poorly defined. We previously reported that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is crucial for diverting fatty acids (FAs) from membrane lipid synthesis to TAG and thereby protecting against FA-induced cell death in leaves. Here, we show that overexpression of PDAT1 enhances the turnover of FAs in leaf lipids. Using the trigalactosyldiacylglycerol1-1 (tgd1-1) mutant, which displays substantially enhanced PDAT1-mediated TAG synthesis, we demonstrate that disruption of SUGAR-DEPENDENT1 (SDP1) TAG lipase or PEROXISOMAL TRANSPORTER1 (PXA1) severely decreases FA turnover, leading to increases in leaf TAG accumulation, to 9% of dry weight, and in total leaf lipid, by 3-fold. The membrane lipid composition of tgd1-1 sdp1-4 and tgd1-1 pxa1-2 double mutants is altered, and their growth and development are compromised. We also show that two Arabidopsis thaliana lipin homologs provide most of the diacylglycerol for TAG synthesis and that loss of their functions markedly reduces TAG content, but with only minor impact on eukaryotic galactolipid synthesis. Collectively, these results show that Arabidopsis lipins, along with PDAT1 and SDP1, function synergistically in directing FAs toward peroxisomal ß-oxidation via TAG intermediates, thereby maintaining membrane lipid homeostasis in leaves.

Analysis of oil droplets in microalgae.

Microalgae are diverse groups of eukaryotic organisms capable of efficiently converting sunlight into chemical energy through photosynthesis with carbohydrates and oils as major storage products. Like other eukaryotes, microalgae store oils in dynamic subcellular organelles named oil droplets. In this chapter, we present a detailed description of basic procedures that can be followed for the isolation of mutants defective in oil droplet biogenesis and for the imaging and analysis of oil droplets in the model unicellular green alga Chlamydomonas reinhardtii. Several commonly used methods for isolating and purifying oil droplets in microalgae are discussed.

Phospholipid:diacylglycerol acyltransferase-mediated triacylglycerol biosynthesis is crucial for protection against fatty acid-induced cell death in growing tissues of Arabidopsis.

Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1-1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1-1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo ¹4C-acetate labeling experiments showed that, compared with wild-type, tgd1-1 exhibits a 3.8-fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over-expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1-1. We also show that detached leaves of both pdat1-2 and tgd1-1 pdat1-2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA-induced cell death in fast-growing tissues of plants.

Dual role for phospholipid:diacylglycerol acyltransferase: enhancing fatty acid synthesis and diverting fatty acids from membrane lipids to triacylglycerol in Arabidopsis leaves.

There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that phospholipid:diacylglycerol acyltransferase1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway. In the trigalactosyldiacylglycerol1-1 mutant, which is defective in eukaryotic thylakoid lipid synthesis, the combined overexpression of PDAT1 with oleosin increases leaf TAG content to 8.6% of the dry weight and total leaf lipid by fourfold. In the plastidic glycerol-3-phosphate acyltransferase1 mutant, which is defective in the prokaryotic glycerolipid pathway, PDAT1 overexpression enhances TAG content at the expense of thylakoid membrane lipids, leading to defects in chloroplast division and thylakoid biogenesis. Collectively, these results reveal a dual role for PDAT1 in enhancing fatty acid and TAG synthesis in leaves and suggest that increasing FAS is the key to engineering high levels of TAG accumulation in green biomass.

Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii.

Microalgal oils have attracted much interest as potential feedstocks for renewable fuels, yet our understanding of the regulatory mechanisms controlling oil biosynthesis and storage in microalgae is rather limited. Using Chlamydomonas reinhardtii as a model system, we show here that starch, rather than oil, is the dominant storage sink for reduced carbon under a wide variety of conditions. In short-term treatments, significant amounts of oil were found to be accumulated concomitantly with starch only under conditions of N starvation, as expected, or in cells cultured with high acetate in otherwise standard growth medium. Time-course analysis revealed that oil accumulation under N starvation lags behind that of starch and rapid oil synthesis occurs only when carbon supply exceeds the capacity of starch synthesis. In the starchless mutant BAFJ5, blocking starch synthesis results in significant increases in the extent and rate of oil accumulation. In the parental strain, but not the starchless mutant, oil accumulation under N starvation was strictly dependent on the available external acetate supply and the amount of oil increased steadily as the acetate concentration increased to the levels several-fold higher than that of the standard growth medium. Additionally, oil accumulation under N starvation is saturated at low light intensities and appears to be largely independent of de novo protein synthesis. Collectively, our results suggest that carbon availability is a key metabolic factor controlling oil biosynthesis and carbon partitioning between starch and oil in Chlamydomonas.

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803.

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K (m) value of the recombinant protein for glutamine was 26.6 +/- 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na(+). Moreover, the K (m) value was decreased to 12.2 +/- 1.9 mmol/L in the presence of Na(+). These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na(+) through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Deltaslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Deltaslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Deltaslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Deltaslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Deltaslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Deltaslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.

A novel ABA-hypersensitive mutant in Arabidopsis defines a genetic locus that confers tolerance to xerothermic stress.

Hot and dry air (harmattan or xerothermic climate) greatly inhibits plant growth, particularly flowering and seed setting of crops. Little is known about the mechanism of plant response to this extreme environmental stress due to the lack of valuable genetic resource. Here, we report the isolation and characteristics of a unique Arabidopsis mutant, hat1 (harmattan tolerant 1), which shows high tolerance to hot and dry air. Under normal growth conditions, the mutant does not differ in morphology and soil drought tolerance compared to the wild type. When subjected to high temperature (42 degrees C) and low humidity (10-15%), however, it could survive up to 6 days, while the wild type (Col-0) died after 24 h. The hat1 mutant also exhibits enhanced tolerance to soil drought, but only under xerothermic conditions. Mutant plants tightly close their stomata to retain water under xerothermic stress, and are more tolerant to high salinity at all developmental stages, accumulating less Na+ and more K+ than wild-type plants during NaCl treatment. Interestingly, hat1 plants are also ABA-hypersensitive. Genetic analysis revealed that the hat1 phenotype is caused by a dominant mutation at a single nuclear locus. Mapping studies indicate that Hat1 is located at an interval of 168 kb on chromosome 5 in which 21 genes are known to be regulated by diverse abiotic stresses. A mutant of this kind, to our knowledge, has not been previously reported. Thus, this report serves as a starting point in the genetic dissection of the plant response to xerothermic stress, and provides physiological and genetic evidence of the existence of a novel abiotic stress response pathway that is also ABA-dependent.