Salinity is a serious limiting factor for the growth of rhizobia. Some rhizobia are tolerant to salt stress and promote plant growth, but the mechanisms underlying these effects are poorly characterized. The growth responses and osmoprotectants in four Bradyrhizobium strains were examined under salt stress in this study. Two-dimensional electrophoresis (2-DE) and mass spectrometry were conducted to investigate protein profiles in rhizobia exposed to salt stress. Subsequently, salt tolerance in stylo (Stylosanthes guianensis) inoculated with rhizobia was further detected in hydroponics. Results showed that the Bradyrhizobium strain RJS9-2 exhibited higher salt tolerance than the other three Bradyrhizobium strains. RJS9-2 was able to grow at 0.35 M NaCl treatment, while the other three Bradyrhizobium strains did not grow at 0.1 M NaCl treatment. Salt stress induced IAA production, and accumulation of proline, betaine, ectoine, and trehalose was observed in RJS9-2 but not in PN13-1. Proteomics analysis identified 14 proteins regulated by salt stress in RJS9-2 that were mainly related to the ABC transporter, stress response, and protein metabolism. Furthermore, under saline conditions, the nodule number, plant dry weight, and N concentration in stylo plants inoculated with RJS9-2 were higher than those in plants inoculated with PN13-1. These results suggest that the tolerance of RJS9-2 to salt stress may be achieved by the coordination of indole-3-acetic acid (IAA) production, osmoprotectant accumulation, and protein expression, thus promoting stylo growth. Full article

ADP-glucose pyrophosphorylase (AGPase) is the first rate-limiting enzyme in starch biosynthesis and plays crucial roles in multiple biological processes. Despite its importance, AGPase is poorly studied in starchy fruit crop banana (Musa acuminata L.). In this study, eight MaAGPase genes have been identified genome-wide in M. acuminata, which could be clustered into the large (APL) and small (APS) subunits. Comprehensive transcriptomic analysis revealed temporal and spatial expression variations of MaAPLs and MaAPSs and their differential responses to abiotic/biotic stresses in two banana genotypes, Fen Jiao (FJ) and BaXi Jiao (BX). MaAPS1 showed generally high expression at various developmental and ripening stages and in response to abiotic/biotic stresses in both genotypes. MaAPL-3 and -2a were specifically induced by abiotic stresses including cold, salt, and drought, as well as by fungal infection in FJ, but not in BX. The presence of hormone-related and stress-relevant cis-acting elements in the promoters of MaAGPase genes suggests that MaAGPases may play an important role in multiple biological processes. Taken together, this study provides new insights into the complex transcriptional regulation of AGPases, underlying their key roles in promoting starch biosynthesis and enhancing stress tolerance in banana. Full article

The BRS1 (BRI1 Suppressor 1) gene encodes a serine carboxypeptidase that plays a critical role in the brassinosteroid signaling pathway. However, its specific biological function remains unclear. In this study, the developmental role of BRS1 was investigated in Arabidopsis thaliana. We found that overexpressing BRS1 resulted in significantly more lateral roots in different Arabidopsis ecotypes (WS2 and Col-0) and in brassinosteroid mutants (bri1-5 and det2-28). Further research showed that BRS1 facilitates the process whereby lateral root primordia break through the endodermis, cortex, and epidermis. Consistent with this, BRS1 was found to be highly expressed in the root endodermis and accumulated in the extracellular space around the dome of the lateral root primordia. Taken together, these results highlight the role of BRS1 in the process of lateral root emergence and provide new insight into the role of serine carboxypeptidases in plant root development. Full article

PKA (protein lysine acetylation) is a critical post-translational modification that regulates various developmental processes, including seed development. However, the acetylation events and dynamics on a proteomic scale in this process remain largely unknown, especially in rice early seed development. We report the first quantitative acetylproteomic study focused on rice early seed development by employing a mass spectral-based (MS-based), label-free approach. A total of 1817 acetylsites on 1688 acetylpeptides from 972 acetylproteins were identified in pistils and seeds at three and seven days after pollination, including 268 acetyproteins differentially acetylated among the three stages. Motif-X analysis revealed that six significantly enriched motifs, such as (DxkK), (kH) and (kY) around the acetylsites of the identified rice seed acetylproteins. Differentially acetylated proteins among the three stages, including adenosine diphosphate (ADP) -glucose pyrophosphorylases (AGPs), PDIL1-1 (protein disulfide isomerase like 1-1), hexokinases, pyruvate dehydrogenase complex (PDC) and numerous other regulators that are extensively involved in the starch and sucrose metabolism, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle and photosynthesis pathways during early seed development. This study greatly expanded the rice acetylome dataset, and shed novel insight into the regulatory roles of PKA in rice early seed development. Full article

Ascorbate oxidase (AO) plays an important role in cell growth through the modulation of reduction/oxidation (redox) control of the apoplast. Here, a cotton (Gossypium hirsutum) apoplastic ascorbate oxidase gene (GhAO1) was obtained from fast elongating fiber tissues. GhAO1 belongs to the multicopper oxidase (MCO) family and includes a signal peptide and several transmembrane regions. Analyses of quantitative real-time polymerase chain reaction (QRT-PCR) and enzyme activity showed that GhAO1 was expressed abundantly in 15-day post-anthesis (dpa) wild-type (WT) fibers in comparison with fuzzless-lintless (fl) mutant ovules. Subcellular distribution analysis in onion cells demonstrated that GhAO1 is localized in the cell wall. In transgenic tobacco bright yellow-2 (BY-2) cells with ectopic overexpression of GhAO1, the enhancement of cell growth with 1.52-fold increase in length versus controls was indicated, as well as the enrichment of both total ascorbate in whole-cells and dehydroascorbate acid (DHA) in apoplasts. In addition, promoted activities of AO and monodehydroascorbate reductase (MDAR) in apoplasts and dehydroascorbate reductase (DHAR) in whole-cells were displayed in transgenic tobacco BY-2 cells. Accumulation of H₂O₂, and influenced expressions of Ca²⁺ channel genes with the activation of NtMPK9 and NtCPK5 and the suppression of NtTPC1B were also demonstrated in transgenic tobacco BY-2 cells. Finally, significant induced expression of the tobacco NtAO gene in WT BY-2 cells under indole-3-acetic acid (IAA) treatment appeared; however, the sensitivity of the NtAO gene expression to IAA disappeared in transgenic BY-2 cells, revealing that the regulated expression of the AO gene is under the control of IAA. Taken together, these results provide evidence that GhAO1 plays an important role in fiber cell elongation and may promote cell growth by generating the oxidation of apoplasts, via the auxin-mediated signaling pathway. Full article

Phosphorus (P) is an important mineral nutrient for plant growth and development. Overexpressing AtWRKY6 (35S:WRKY6-9) was more sensitive and wrky6 (wrky6-1) was more resistant under low Pi conditions. To better understand the function of AtWRKY6 under low phosphate stress conditions, we applied two-dimensional gel electrophoresis (2-DE) to analyse differentially expressed proteins in the shoots and roots between wild type, 35S:WRKY6-9 and wrky6-1 after phosphorus deficiency treatment for three days. The results showed 88 differentially abundant protein spots, which were identified between the shoots and roots of 35S:WRKY6-9 and wrky6-1 plants. In addition, 59 differentially expressed proteins were identified in the leaves and roots of 35S:WRKY6-9 plants. After analysis, 9 genes with W-box elements in their promoter sequences were identified in the leaves, while 6 genes with W-box elements in their promoter sequences were identified in the roots. A total of 8 genes were identified as potential target genes according to the quantitative PCR (QPCR) and two dimension difference gel electrophoresis, (2D-DIGE) results, including ATP synthase, gln synthetase, nitrilase, 14-3-3 protein, carbonic anhydrases 2, and tryptophan synthase. These results provide important information concerning the AtWRKY6 regulation network and reveal potential vital target genes of AtWRKY6 under low phosphorus stress. two dimension difference gel electrophoresis, 2D-DIGE Full article

Roots of Arabidopsis thaliana seedlings grown in the laboratory using the traditional plant-growing culture system (TPG) were covered to maintain them in darkness. This new method is based on a dark chamber and is named the improved plant-growing method (IPG). We measured the light conditions in dark chambers, and found that the highest light intensity was dramatically reduced deeper in the dark chamber. In the bottom and side parts of dark chambers, roots were almost completely shaded. Using the high-throughput RNA sequencing method on the whole RNA extraction from roots, we compared the global gene expression levels in roots of seedlings from these two conditions and identified 141 differently expressed genes (DEGs) between them. According to the KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment, the flavone and flavonol biosynthesis and flavonoid biosynthesis pathways were most affected among all annotated pathways. Surprisingly, no genes of known plant photoreceptors were identified as DEGs by this method. Considering that the light intensity was decreased in the IPG system, we collected four sections (1.5 cm for each) of Arabidopsis roots grown in TPG and IPG conditions, and the spatial-related differential gene expression levels of plant photoreceptors and polar auxin transporters, including CRY1, CRY2, PHYA, PHYB, PHOT1, PHOT2, and UVR8 were analyzed by qRT-PCR. Using these results, we generated a map of the spatial-related expression patterns of these genes under IPG and TPG conditions. The expression levels of light-related genes in roots is highly sensitive to illumination and it provides a background reference for selecting an improved culture method for laboratory-maintained Arabidopsis seedlings. Full article

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF_138,175,258 and SRPP_117,204,243, were characterized from Hevea brasiliensis Reyan (RY) 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF₂₅₈ has a β subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE), each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF₁₇₅ or REF₂₅₈ were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis. Full article

Plant anthocyanin biosynthesis is well understood, but the regulatory mechanism in purple foliage tea remains unclear. Using isobaric tag for relative and absolute quantification (iTRAQ), 815 differential proteins were identified in the leaves of Zijuan tea, among which 20 were associated with the regulation of anthocyanin metabolism. We found that the abundances of anthocyanin synthesis-related enzymes such as chalcone synthase, chalcone isomerase, dihydroflavonol 4-reductase and anthocyanin synthetase, as well as anthocyanin accumulation-related UDP-glucosyl transferase and ATP-binding cassette (ABC) transporters in the purple leaves were all significantly higher than those in the green leaves. The abundances of the transcription factors bHLH and HY5, regulating anthocyanin biosynthesis at transcriptional level were also obviously higher in purple leaves than those in green leaves. In addition, bifunctional 3-dehydroquinate dehydratase and chorismate mutase in purple leaves were distinctly higher in abundance compared to green leaves, which provided sufficient phenylalanine substrate for anthocyanin synthesis. Furthermore, lignin synthesis was found to be reduced due to the lower abundances of cinnamoyl-CoA reductase 1, peroxidase 15 and laccase-6, which resulted in increase of intermediates flow into anthocyanin synthesis pathway. The physiological data were consistent with proteomic results. These four aspects of biosynthetic regulation contribute to anthocyanin accumulation in purple leaves of Zijuan tea. Full article

The sugar beet monosomic addition line M14 is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and shows tolerance to salt stress. Our study focuses on exploring the molecular mechanism of the salt tolerance of the sugar beet M14. In order to identify differentially expressed genes in M14 under salt stress, a subtractive cDNA library was generated by suppression subtractive hybridization (SSH). A total of 36 unique sequences were identified in the library and their putative functions were analyzed. One of the genes, S-adenosylmethionine synthetase (SAMS), is the key enzyme involved in the biosynthesis of S-adenosylmethionine (SAM), a precursor of polyamines. To determine the potential role of SAMS in salt tolerance, we isolated BvM14-SAMS2 from the salt-tolerant sugar beet M14. The expression of BvM14-SAMS2 in leaves and roots was greatly induced by salt stress. Overexpression of BvM14-SAMS2 in Arabidopsis resulted in enhanced salt and H₂O₂ tolerance. Furthermore, we obtained a knock-down T-DNA insertion mutant of AtSAMS3, which shares the highest homology with BvM14-SAMS2. Interestingly, the mutant atsam3 showed sensitivity to salt and H₂O₂ stress. We also found that the antioxidant system and polyamine metabolism play an important role in salt and H₂O₂ tolerance in the BvM14-SAMS2-overexpressed plants. To our knowledge, the function of the sugar beet SAMS has not been reported before. Our results have provided new insights into SAMS functions in sugar beet. Full article

Dianthus spiculifolius, a perennial herbaceous flower and a member of the Caryophyllaceae family, has strong resistance to cold and drought stresses. To explore the transcriptional responses of D. spiculifolius to individual and combined stresses, we performed transcriptome sequencing of seedlings under normal conditions or subjected to cold treatment (CT), simulated drought treatment (DT), or their combination (CTDT). After de novo assembly of the obtained reads, 112,015 unigenes were generated. Analysis of differentially expressed genes (DEGs) showed that 2026, 940, and 2346 genes were up-regulated and 1468, 707, and 1759 were down-regulated in CT, DT, and CTDT samples, respectively. Among all the DEGs, 182 up-regulated and 116 down-regulated genes were identified in all the treatment groups. Analysis of metabolic pathways and regulatory networks associated with the DEGs revealed overlaps and cross-talk between cold and drought stress response pathways. The expression profiles of the selected DEGs in CT, DT, and CTDT samples were characterized and confirmed by quantitative RT-PCR. These DEGs and metabolic pathways may play important roles in the response of D. spiculifolius to the combined stress. Functional characterization of these genes and pathways will provide new targets for enhancement of plant stress tolerance through genetic manipulation. Full article

Salinity is a severe abiotic stress limiting agricultural yield and productivity. Plants have evolved various strategies to cope with salt stress. Chloroplasts are important photosynthesis organelles, which are sensitive to salinity. An understanding of molecular mechanisms in chloroplast tolerance to salinity is of great importance for genetic modification and plant breeding. Previous studies have characterized more than 53 salt-responsive genes encoding important chloroplast-localized proteins, which imply multiple vital pathways in chloroplasts in response to salt stress, such as thylakoid membrane organization, the modulation of photosystem II (PS II) activity, carbon dioxide (CO₂) assimilation, photorespiration, reactive oxygen species (ROS) scavenging, osmotic and ion homeostasis, abscisic acid (ABA) biosynthesis and signaling, and gene expression regulation, as well as protein synthesis and turnover. This review presents an overview of salt response in chloroplasts revealed by gene characterization efforts. Full article