Transcriptome analysis reveals how cadmium promotes root development and accumulates in Apocynum venetum, a promising plant for greening cadmium-contaminated soil.

Cadmium (Cd) contamination poses a substantial threat the environment, necessitating effective remediation strategies. Phytoremediation emerges as a cost-efficient and eco-friendly approach for reducing Cd levels in the soil. In this study, the suitability of A. venetum for ameliorating Cd-contaminated soils was evaluated. Mild Cd stress promoted seedling and root growth, with the root being identified as the primary tissue for Cd accumulation. The Cd content of roots ranged from 0.35 to 0.55 mg/g under treatment with 10-50 µM CdCl2·2.5 H2O, and the bioaccumulation factor ranged from 28.78 to 84.43. Transcriptome sequencing revealed 20,292 unigenes, and 7507 nonredundant differentially expressed genes (DEGs) were identified across five comparison groups. DEGs belonging to the "MAPK signaling pathway-plant," "monoterpenoid biosynthesis," and "flavonoid biosynthesis pathway" exhibited higher expression levels in roots compared to stems and leaves. In addition, cytokinin-related DEGs, ROS scavenger genes, such as P450, glutathione-S-transferase (GST), and superoxide dismutase (SOD), and the cell wall biosynthesis-related genes, CSLG and D-GRL, were also upregulated in the root tissue, suggesting that Cd promotes root development. Conversely, certain ABC transporter genes, (e.g, NRAMP5), and some vacuolar iron transporters, predominantly expressed in the roots, displayed a strong correlation with Cd content, revealing the mechanism underlying the compartmentalized storage of Cd in the roots. KEGG enrichment analysis of DEGs showed that the pathways associated with the biosynthesis of flavonoids, lignin, and some terpenoids were significantly enriched in the roots under Cd stress, underscoring the pivotal role of these pathways in Cd detoxification. Our study suggests A. venetum as a potential Cd-contaminated phytoremediation plant and provides insights into the molecular-level mechanisms of root development promotion and accumulation mechanism in response to Cd stress.

Effect of planting salt-tolerant legumes on coastal saline soil nutrient availability and microbial communities.

Soil salinization is a serious global environmental problem affecting sustainable development of agriculture. Legumes are excellent candidates for the phytoremediation of saline soils; however, how soil microbes mediate the amelioration of coastal saline ecosystems is unknown. In this study, two salt-tolerant legumes, Glycine soja and Sesbania cannabina were planted in coastal saline soil for three years. Soil nutrient availability and microbiota structure (including bacteria, fungi, and diazotrophs) were compared between the phytoremediated soils and control soil (barren land). Planting legumes reduced soil salinity, and increased total carbon, total nitrogen, and NO3--N contents. Among the soil microbiota, some nitrogen-fixing bacteria (e.g., Azotobacter) were enriched in legumes, which were probably responsible for soil nitrogen accumulation. The complexity of the bacterial, fungal, and diazotrophic networks increased significantly from the control to the phytoremediated soils, suggesting that the soil microbial community formed closer ecological interactions during remediation. Furthermore, the dominant microbial functions were chemoheterotrophy (24.75%) and aerobic chemoheterotrophy (21.97%) involved in the carbon cycle, followed by nitrification (13.68%) and aerobic ammonia oxidation (13.34%) involved in the nitrogen cycle. Overall, our findings suggested that G. soja and S. cannabina legumes were suitable for ameliorating saline soils as they decreased soil salinity and increased soil nutrient content, with microorganisms especially nitrogen-fixing bacteria, playing an important role in this remediation process.

Heterologous overexpression of Apocynum venetum flavonoids synthetase genes improves Arabidopsis thaliana salt tolerance by activating the IAA and JA biosynthesis pathways.

Salt stress is a serious abiotic stress that primarily inhibits plant growth, resulting in severe yield losses. Our previous research found that flavonoids play important roles in A. venetum salt stress tolerance. In response to salt stress, we noted that the flavonoid content was depleted in A. venetum. However, the detailed mechanism is still not clear. In this study, the expression patterns of three flavonoids synthetase genes, AvF3H, AvF3'H, and AvFLS were systemically analyzed under salt stress in A. venetum seedlings. The salt tolerance of transgenic Arabidopsis plants was improved by heterologous overexpression of these synthetase genes. The NBT and DAB staining results as well as H2O2 and O2•- content analysis revealed that under salt stress, ROS molecules were reduced in transgenic plants compared to WT plants, which corresponded to the activation of the antioxidant enzyme system and an increase in total flavonoid content, particularly rutin, eriodictyol, and naringerin in transgenic plants. External application of flavonoids reduced ROS damage in WT plants just like what we observed in the transgenic plants (without the external application). Additionally, our transcriptome analysis demonstrated that auxin and jasmonic acid biosynthesis genes, as well as signaling transduction genes, were primarily activated in transgenic plants under salt stress, leading to activation of the cell wall biosynthesis or modification genes that promote plant growth. As a result, we investigated the mechanism through flavonoids enhance the salt tolerance, offering a theoretical foundation for enhancing salt tolerance in plants.

Using CRISPR-Cas9 Technology to Eliminate Xyloglucan in Tobacco Cell Walls and Change the Uptake and Translocation of Inorganic Arsenic.

Xyloglucan is a quantitatively major polysaccharide in the primary cell walls of flowering plants and has been reported to affect plants' ability to tolerate toxic elements. However, it is not known if altering the amounts of xyloglucan in the wall influences the uptake and translocation of inorganic arsenic (As). Here, we identified two Nicotiana tabacum genes that encode xyloglucan-specific xylosyltransferases (XXT), which we named NtXXT1 and NtXXT2. We used CRISPR-Cas9 technology to generate ntxxt1, ntxxt2, and ntxxt1/2 mutant tobacco plants to determine if preventing xyloglucan synthesis affects plant growth and their ability to accumulate As. We show that NtXXT1 and NtXXT2 are required for xyloglucan biosynthesis because no discernible amounts of xyloglucan were present in the cell walls of the ntxxt1/2 double mutant. The tobacco double mutant (ntxxt1/2) and the corresponding Arabidopsis mutant (atxxt1/2) do not have severe growth defects but do have a short root hair phenotype and a slow growth rate. This phenotype is rescued by overexpressing NtXXT1 or NtXXT2 in atxxt1/2. Growing ntxxt mutants in the presence of AsIII or AsV showed that the absence of cell wall xyloglucan affects the accumulation and translocation of As. Most notably, root retention of As increased substantially and the amounts of As translocated to the shoots decreased in ntxxt1/2. Our results suggest that xyloglucan-deficient plants provide a strategy for the phytoremediation of As contaminated soils.

Genomic insights on fighting bacterial wilt by a novel Bacillus amyloliquefaciens strain Cas02.

Bacterial wilt, caused by the Ralstonia solanacearum, can infect several economically important crops. However, the management strategies available to control this disease are limited. Plant growth-promoting rhizobacteria (PGPR) have been considered promising biocontrol agents. In this study, Bacillus amyloliquefaciens strain Cas02 was isolated from the rhizosphere soil of healthy tobacco plants and evaluated for its effect on plant growth promotion and bacterial wilt suppression. Strain Cas02 exhibited several growth-promoting-related features including siderophore production, cellulase activity, protease activity, ammonia production and catalase activity. Moreover, strain Cas02 showed a significant inhibitory growth effect on R. solanacearum, and its active substances were separated and identified to be macrolactin A and macrolactin W by HPLC-DAD-ESI-MS/MS. Both greenhouse and field experiments demonstrated a good performance of Cas02 in plant growth promotion and bacterial wilt suppression. To explore the underlying genetic mechanisms, complete genome sequencing was performed and the gene clusters responsible for antibacterial metabolites expression were identified. Overall, these findings suggest that the strain Cas02 could be a potential biocontrol agent in bacterial wilt management and a source of antimicrobial compounds for further exploitation.

Patterns in the Microbial Community of Salt-Tolerant Plants and the Functional Genes Associated with Salt Stress Alleviation.

Salinity is an important abiotic stress affecting plant growth. We have known that plants can recruit beneficial microbes from the surrounding soil. However, the ecological functions of the core microbiome in salt-tolerant plants, together with their driving factors, remain largely unexplored. Here, we employed both amplicon and shotgun metagenomic sequencing to investigate the microbiome and function signatures of bulk soil and rhizocompartment samples from three salt-tolerant plants (legumes Glycine soja and Sesbania cannabina and nonlegume Sorghum bicolor). Strong filtration effects for microbes and functional genes were found in the rhizocompartments following a spatial gradient. The dominant bacteria belonged to Ensifer for legumes and Bacillus for S. bicolor. Although different salt-tolerant plants harbored distinct bacterial communities, they all enriched genes involved in cell motility, Na+ transport, and plant growth-promoting function (e.g., nitrogen fixation and phosphate solubilization) in rhizoplane soils, implying that the microbiome assembly of salt-tolerant plants might depend on the ecological functions of microbes rather than microbial taxa. Moreover, three metagenome-assembled genomes affiliated to Ensifer were obtained, and their genetic basis for salt stress alleviation were predicted. Soil pH, electrical conductivity, and total nitrogen were the most important driving factors for explaining the above microbial and functional gene selection. Correspondingly, the growth of an endophyte, Ensifer meliloti CL09, was enhanced by providing root exudates, suggesting that root exudates might be one of factors in the selection of rhizosphere and endosphere microbiota. Overall, this study reveals the ecological functions of the populations inhabiting the root of salt-tolerant plants. IMPORTANCE Salinity is an important but little-studied abiotic stressor affecting plant growth. Although several previous reports have examined salt-tolerant plant microbial communities, we still lack a comprehensive understanding about the functional characteristics and genomic information of this population. The results of this study revealed the root-enriched and -depleted bacterial groups, and found three salt-tolerant plants harbored different bacterial populations. The prediction of three metagenome-assembled genomes confirmed the critical role of root dominant species in helping plants tolerate salt stress. Further analysis indicated that plants enriched microbiome from soil according to their ecological functions but not microbial taxa. This highlights the importance of microbial function in enhancing plant adaptability to saline soil and implies that we should pay more attention to microbial function and not only to taxonomic information. Ultimately, these results provide insight for future agriculture using the various functions of microorganisms on the saline soil.

Comparative transcriptome analysis reveals the molecular mechanism of salt tolerance in Apocynum venetum.

Apocynum venetum is a traditional Chinese medicinal herb with tolerance to various abiotic stresses, especially, salinity. However, only a few studies have investigated the salt-tolerant mechanism of this non-halophyte under salt stress at phenotypic and physiological levels. To explore the molecular mechanism of salinity tolerance in A. venetum, the global transcriptome profiles of seedling leaves under different salt-stress durations, using 200 mM NaCl, were analyzed. De novo assembly of approximately 715 million high-quality reads and approximately 105.61 Gb sequence data was performed. In total, 2822 differentially expressed genes (DEGs) were identified. DEGs were significantly enriched in flavonoid metabolism-related pathways such as "flavonoid biosynthesis" and "phenylpropanoid biosynthesis". Most of these DEGs were downregulated under salt stress. However, genes encoding the non-selective cation channels and antioxidants were upregulated under salt stress, whereas most cell wall-related DEGs were downregulated. Consequently, the concentration of flavonoids decreased, whereas that of Na+ increased with exposure time. Thus, we hypothesized that the accumulation of Na+ in the leaves, which resulted in reduced flavonoid concentration under salt stress, directly led to a decrease in the salt tolerance of A. venetum. This was verified by overexpressing four flavonoid synthesis pathway genes in Arabidopsis. The transgenic plants showed higher salt tolerance than the wild-type plants due to the accumulation of total flavonoids. These physiological and transcriptome analyses of A. venetum revealed major molecular underpinnings contributing to the responses of A. venetum to salt stress, thereby improving our understanding of the molecular mechanisms underlying salt tolerance in A. venetum and plants in general. The findings serve as a basis for functional studies on and engineering strategies for plant salinity tolerance.

Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca's response to salinity.

Quantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

Overexpression of an Apocynum venetum flavonols synthetase gene confers salinity stress tolerance to transgenic tobacco plants.

Soil salinity is a major limiting factor for agricultural production, threatening food security worldwide. A thorough understanding of the mechanisms underlying plant responses is required to effectively counter its deleterious effects on crop productivity. Total flavonoid accumulation reportedly improves salinity tolerance in many crops. Therefore, we isolated the full-length cDNA of a flavonol synthetase (FLS) gene from Apocynum venetum (AvFLS). The gene contained a 1008-bp open reading frame encoding a protein composed of 335 amino acid residues. Multiple sequence alignment showed that the AvFLS protein was highly homologous to FLSs from other plants. AvFLS was expressed in leaves, stems, roots, flowers, and germinated seeds. Expression pattern analysis revealed that AvFLS was significantly induced by salinity stress. AvFLS overexpression in tobacco positively affected the development and growth of transgenic plants under salinity stress: root and seedling growth were inhibited to a lesser extent, while seed germination rate increased. Additionally, the overexpression of AvFLS under salinity stress resulted in an increase in total flavonoid content (1.63 mg g-1 in wild-type samples and 4.63 mg g-1 on average in transgenic samples), which accompanied the increase in the activity of antioxidant enzymes and inhibited the production of reactive oxygen species. Further, AvFLS-overexpressing transgenic tobacco plants absorbed more K+ than wild type plants, leading to an increased K+/Na+ ratio, which in turn contributed to the maintenance of Na+/K+ homeostasis. These findings suggest that an AvFLS-induced increase in total flavonoid content enhanced plant salinity tolerance, implying the importance of AvFLS gene responses to salinity stress.

ERF4 and MYB52 transcription factors play antagonistic roles in regulating homogalacturonan de-methylesterification in Arabidopsis seed coat mucilage.

Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other's DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4-MYB52 transcriptional complex.

Biochar and fertilizer improved the growth and quality of the ice plant (Mesembryanthemum crystallinum L.) shoots in a coastal soil of Yellow River Delta, China.

Coastal soil is an important land reserve that may be used to alleviate the shortage of cultivated land; however, this soil is stressed by saline conditions and nutrient deficiency. Biochar offers the potential to reclaim coastal soil, but the response of plant growth to biochar addition in salt-affected soil is species-dependent. In this study, the response of ice plant (Mesembryanthemum crystallinum L.), an economically valuable halophyte that grows in the coastal soil of the Yellow River Delta, to wood chip biochar (WBC) either alone or in combination with chemical fertilizer was investigated using a 90-day pot experiment. The WBC enhanced the growth of ice plants in the coastal soil, but combining it with chemical fertilizer did not increase its effect. The nutritional quality of the plants was improved by the addition of WBC, regardless of whether chemical fertilizer was applied; moreover, WBC amendment enhanced photosynthesis and reduced the oxidative stress of the plants. The ameliorated soil properties (e.g., soil organic matter and water holding capacity) and increased contents of available macronutrients (e.g., P and K) and micronutrients (e.g., Mg, Mn, B and Zn) resulting from soil amendment with WBC may have contributed to the enhanced growth and quality of the ice plants. Additionally, in soil modified with WBC, an increased abundance of beneficial taxa (e.g., Erythrobacter, Sphingomonas and Lysobacter) and a shift in the microbial community may also have helped to improve the growth and quality of the ice plants. The results of our study provide useful information for developing a biochar-based technology to use in combination with valuable halophytes to reclaim degraded coastal soil and enhance food security.

Identification of two functional xyloglucan galactosyltransferase homologs BrMUR3 and BoMUR3 in brassicaceous vegetables.

Xyloglucan (XyG) is the predominant hemicellulose in the primary cell walls of most dicotyledonous plants. Current models of these walls predict that XyG interacts with cellulose microfibrils to provide the wall with the rigidity and strength necessary to maintain cell integrity. Remodeling of this network is required to allow cell elongation and plant growth. In this study, homologs of Arabidopsis thaliana MURUS3 (MUR3), which encodes a XyG-specific galactosyltransferase, were obtained from Brassica rapa (BrMUR3) to Brassica oleracea (BoMUR3). Genetic complementation showed that BrMUR3 and BoMUR3 rescue the phenotypic defects of the mur3-3 mutant. Xyloglucan subunit composition analysis provided evidence that BrMUR3 and BoMUR3 encode a galactosyltransferase, which transfers a galactose residue onto XyG chains. The detection of XXFG and XLFG XyG subunits (restoration of fucosylated side chains) in mur3-3 mutants overexpressing BrMUR3 or BoMUR3 show that MUR3 from Brassica to Arabidopsis are comparable as they add Gal to the third xylosyl residue of the XXXG subunit. Our results provide additional information for functional dissection and evolutionary analysis of MUR3 genes derived from brassicaceous species.

Beneficial effects of polysaccharide-rich extracts from Apocynum venetum leaves on hypoglycemic and gut microbiota in type 2 diabetic mice.

Diabetes is one of the most concerned metabolic diseases worldwide and threaten public health. In the present work, two polysaccharide-rich extracts from Apocynum venetum leaves were extracted using distilled water and alkaline solution (0.05 M NaOH), and fully characterized. Hypoglycemic and hypolipidemic effects of two polysaccharide-rich extracts on high-fat diet and streptozocin-induced type 2 diabetic mice were investigated. Treatment of alkaline extracted polysaccharide-rich products significantly decreased the levels of fasting blood glucose, serum insulin, glycated serum protein, as well as serum lipids profiles including total cholesterol, triacylglycerols, low-density lipoprotein cholesterol, and nonesterified fatty acid. Meanwhile, the reduced glycogen contents in liver were prominently improved, and the oxidative damage were markedly ameliorated by alkaline extracted polysaccharide products in diabetic mice. Furthermore, both polysaccharide-rich extracts could reverse the gut microbiota dysbiosis in diabetic mice by increasing the abundance of genera Odoribacter, Anaeroplasma, Parasutterella, and Muribaculum; while by decreasing the abundance of genera Enterococcus, Klebsiella, and Aerococcus. This study provides new sights for exploitation of Apocynum venetum extracts as a promising anti-diabetic nutraceutical for the treatment of type 2 diabetes and metabolic syndrome.

Overexpression of NtEXPA11 modulates plant growth and development and enhances stress tolerance in tobacco.

Apart from providing the much-needed strength, plant cell walls define the shape, size and function of cells. As such, there is a constant change in the cell wall dynamics. These are facilitated by various enzymes and proteins. Expansins are a typical example of those cell wall proteins that are involved in cell wall modifications underlying many plant developmental and physiological processes. In this work, we investigated the role of NtEXPA11 gene in tobacco by generating transgenic plants ectopically expressing NtEXPA11 under the control of CaMV35S promoter. Gene expression analysis revealed that although this gene was present in all the studied tissues in WT plants, its transcript levels were highest in the stems, flowers and leaves and lowest in the roots. Following its overexpressing in tobacco, the NtEXPA11-OX plants exhibited an enhanced growth phenotype. Compared to WT plants, these plants demonstrated an increased growth rate which was characterized by a vigorous root system as well as an accelerated growth rate during their early developmental stages. NtEXPA11-OX plants also developed significantly bigger leaves and internode lengths. They exhibited a 57% increase (NtEXPA11-2) and 98% increase (NtEXPA11-19) in leaf area when grown on MS media. Most interestingly, NtEXPA11-OX plants had significantly bigger pith and parenchyma cells compared to their WT counterparts. Furthermore, we noted that NtEXPA11 plays an important role in plant adaptation to stresses as indicated by the improved tolerance to drought and salt stress of the NtEXPA11-OX plants compared to the WT plants.

Genome-Wide Identification and Analysis of P-Type Plasma Membrane H+-ATPase Sub-Gene Family in Sunflower and the Role of HHA4 and HHA11 in the Development of Salt Stress Resistance.

The P-type plasma membrane (PM) H+-ATPase plays a major role during the growth and development of a plant. It is also involved in plant resistance to a variety of biotic and abiotic factors, including salt stress. The PM H+-ATPase gene family has been well characterized in Arabidopsis and other crop plants such as rice, cucumber, and potato; however, the same cannot be said in sunflower (Helianthus annuus). In this study, a total of thirteen PM H+-ATPase genes were screened from the recently released sunflower genome database with a comprehensive genome-wide analysis. According to a systematic phylogenetic classification with a previously reported species, the sunflower PM H+-ATPase genes (HHAs) were divided into four sub-clusters (I, II, IV, and V). In addition, systematic bioinformatics analyses such as gene structure analysis, chromosome location analysis, subcellular localization predication, conserved motifs, and Cis-acting elements of promoter identification were also done. Semi-quantitative PCR analysis data of HHAs in different sunflower tissues revealed the specificity of gene spatiotemporal expression and sub-cluster grouping. Those belonging to sub-cluster I and II exhibited wide expression in almost all of the tissues studied while sub-cluster IV and V seldom showed expression. In addition, the expression of HHA4, HHA11, and HHA13 was shown to be induced by salt stress. The transgenic plants overexpressing HHA4 and HHA11 showed higher salinity tolerance compared with wild-type plants. Further analysis showed that the Na+ content of transgenic Arabidopsis plants decreased under salt stress, which indicates that PM H+ ATPase participates in the physiological process of Na+ efflux, resulting in salt resistance of the plants. This study is the first to identify and analyze the sunflower PM H+ ATPase gene family. It does not only lay foundation for future research but also demonstrates the role played by HHAs in salt stress tolerance.

The Root Nodule Microbiome of Cultivated and Wild Halophytic Legumes Showed Similar Diversity but Distinct Community Structure in Yellow River Delta Saline Soils.

Symbiotic associations between leguminous plants and their nodule microbiome play a key role in sustainable agriculture by facilitating the fixation of atmospheric nitrogen and enhancing plant stress resistance. This study aimed to decipher the root nodule microbiome of two halophytic legumes, Sesbania cannabina and Glycine soja, which grow in saline soils of the Yellow River Delta, China, using PacBio's circular consensus sequencing for full-length bacterial 16S rRNA gene to obtain finer taxonomic information. The cultivated legume Glycine max was used for comparison. We identified 18 bacterial genera and 55 species in nodule samples, which mainly classified to Proteobacteria, and rhizobial genus Ensifer was the predominant group. The three legumes showed similarity in operational taxonomic unit (OTU) diversity but distinction in OTU richness, indicating that they harbor similar bacterial species with different relative contents. The results of principal coordinates analysis and ANOSIM tests indicated that G. soja and G. max have similar nodule bacterial communities, and these communities differ from that of S. cannabina. Wild legumes S. cannabina and G. soja both harbored a higher number of rhizobia, while G. max possessed more non-rhizobial bacteria. These differences could be associated with their adaptability to saline-alkali stress and revealed clues on the nodule endophytes with relative importance of culturable rhizobial symbionts.

Tubby-like Protein 2 regulates homogalacturonan biosynthesis in Arabidopsis seed coat mucilage.

KEY MESSAGE: A possible transcription factor TLP2 was identified to be involved in the regulation of HG biosynthesis in Arabidopsis seed mucilage. TLP2 can translocate into nucleus from plasma membrane by interacting with NF-YC3. The discovery of TLP2 gene function can further fulfill the regulatory network of pectin biosynthesis in Arabidopsis thaliana. Arabidopsis seed coat mucilage is an excellent model system to study the biosynthesis, function and regulation of pectin. Rhamnogalacturonan I (RG-I) and homogalacturonan (HG) are the major polysaccharides constituent of the Arabidopsis seed coat mucilage. Here, we identified a Tubby-like gene, Tubby-like protein 2 (TLP2), which was up-regulated in developing siliques when mucilage began to be produced. Ruthenium red (RR) staining of the seeds showed defective mucilage of tlp2-1 mutant after vigorous shaking compared to wild type (WT). Monosaccharide composition analysis revealed that the amount of total sugars and galacturonic acid (GalA) decreased significantly in the adherent mucilage (AM) of tlp2-1 mutant. Immunolabelling and dot immunoblotting analysis showed that unesterified HG decreased in the tlp2-1 mutant. Furthermore, TLP2 can translocate into nucleus by interacting with Nuclear Factor Y subunit C3 (NF-YC3) to function as a transcription factor. RNA-sequence and transactivation assays revealed that TLP2 could activate UDP-glucose 4-epimerase 1 (UGE1). In all, it is concluded that TLP2 could regulate the biosynthesis of HG possibly through the positive activation of UGE1.

Genome-Wide Identification and Expression Profiling Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family in Tobacco (Nicotiana tabacum L.).

Xyloglucan endotransglucosylase/hydrolase genes (XTHs) encode enzymes required for the reconstruction and modification of xyloglucan backbones, which will result in changes of cell wall extensibility during growth. A total of 56 NtXTH genes were identified from common tobacco, and 50 cDNA fragments were verified by PCR amplification. The 56 NtXTH genes could be classified into two subfamilies: Group I/II and Group III according to their phylogenetic relationships. The gene structure, chromosomal localization, conserved protein domains prediction, sub-cellular localization of NtXTH proteins and evolutionary relationships among Nicotiana tabacum, Nicotiana sylvestrisis, Nicotiana tomentosiformis, Arabidopsis, and rice were also analyzed. The NtXTHs expression profiles analyzed by the TobEA database and qRT-PCR revealed that NtXTHs display different expression patterns in different tissues. Notably, the expression patterns of 12 NtXTHs responding to environment stresses, including salinity, alkali, heat, chilling, and plant hormones, including IAA and brassinolide, were characterized. All the results would be useful for the function study of NtXTHs during different growth cycles and stresses.

MYB52 Negatively Regulates Pectin Demethylesterification in Seed Coat Mucilage.

Pectin, which is a major component of the plant primary cell walls, is synthesized and methyl-esterified in the Golgi apparatus and then demethylesterified by pectin methylesterases (PMEs) located in the cell wall. The degree of methylesterification affects the functional properties of pectin, and thereby influences plant growth, development and defense. However, little is known about the mechanisms that regulate pectin demethylesterification. Here, we show that in Arabidopsis (Arabidopsis thaliana) seed coat mucilage, the absence of the MYB52 transcription factor is correlated with an increase in PME activity and a decrease in the degree of pectin methylesterification. Decreased methylesterification in the myb52 mutant is also correlated with an increase in the calcium content of the seed mucilage. Chromatin immunoprecipitation analysis and molecular genetic studies suggest that MYB52 transcriptionally activates PECTIN METHYLESTERASE INHIBITOR6 (PMEI6), PMEI14, and SUBTILISIN-LIKE SER PROTEASE1.7 (SBT1.7) by binding to their promoters. PMEI6 and SBT1.7 have previously been shown to be involved in seed coat mucilage demethylesterification. Our characterization of two PMEI14 mutants suggests that PMEI14 has a role in seed coat mucilage demethylesterification, although its activity may be confined to the seed coat in contrast to PMEI6, which functions in the whole seed. Our demonstration that MYB52 negatively regulates pectin demethylesterification in seed coat mucilage, and the identification of components of the molecular network involved, provides new insight into the regulatory mechanism controlling pectin demethylesterification and increases our understanding of the transcriptional regulation network involved in seed coat mucilage formation.

The tubby-like proteins kingdom in animals and plants.

Each gene of the tubby-like family is characterized by a signature of C-terminal tubby domain. The wide spread of this family in plants and animals implies they have an important function in various organisms. Even though the tubby-like genes are suggested to be putative transcription factors (TFs), how they execute the function as TFs is not yet clear. The biological functions of most animal tubby-like genes have been well studied, especially for vertebrate TUB, TULP1 and TULP3, but not with TULP2 and TULP4. Plants possess more tubby-like genes than animals, but their functions are still elusive except the idea that they are involved in stress responses with indistinct mechanisms. Here we reviewed the current knowledge of the versatile functions and roles of the tubby-like family members in plants and animals.