Seed Maturation Proteins Research Articles

Genome-wide analysis and functional validation reveal the role of late embryogenesis abundant genes in strawberry (Fragaria × ananassa) fruit ripening

BackgroundLate embryogenesis abundant (LEA) proteins play important roles in plant growth and development, as well as stresses responsiveness. Nowadays, it has been found that LEAs also have function in fruit ripening. However, the comprehensive analysis on a genome-wide basis of LEA family remains limited, and the role of LEA in fruit ripening has not been fully explored yet, especially in strawberry, an economic important plant and ideal material for studying fruit ripening.ResultsIn this study, a total of 266 putative LEA proteins were identified and characterized in strawberry genome. Subcellular localization prediction indicated that they were mostly localized in chloroplast, cytoplasm and nucleus. Duplication events detection revealed that whole genome duplication or segmental was the main driver for the expansion of LEA family in strawberry. The phylogenetic analysis suggested that FaLEAs were classified into eight groups, among which, LEA2 was the largest subgroup with 179 members, followed by LEA3, dehydrin (DHN), LEA4 and SMP (seed maturation protein). The LEA1 and DHN groups were speculated to play dominant roles in strawberry fruit development and ripening, according to their larger proportion of members detected as differentially expressed genes during such process. Notably, the expression of FaLEA167 belonging to LEA1 group was altered by strawberry maturation, and inhibited by overexpression of negative regulators of ripening (a cytosolic/plastid glyceraldehyde-3-phosphate dehydrogenase, FaGAPC2 and a cytosolic pyruvate kinase, FaPKc2.2). Subsequently, overexpression of FaLEA167 significantly increased the percentage of fruit at green stage, while reduced the full red fruit proportion. In consistent, the anthocyanins content and the fruit skin color variable reflecting a range from greenness to redness (a* value) were significantly reduced. Whereas, FaLEA167 overexpression apparently up-regulated citric acid, soluble protein and malondialdehyde content, but had no obvious effects on total soluble solids, sugar, flavonoids, phenolics content and antioxidant capacity.ConclusionsThese findings not only provided basic information of FaLEA family for further functional research, but also revealed the involvement of FaLEA167 in negatively regulating strawberry fruit ripening, giving new insights into understanding of FaLEA functions.

Genome-wide identification of late embryogenesis abundant protein family and their key regulatory network in Pinus tabuliformis cold acclimation.

Cold acclimation is a crucial biological process that enables conifers to overwinter safely. The late embryogenesis abundant (LEA) protein family plays a pivotal role in enhancing freezing tolerance during this process. Despite its importance, the identification, molecular functions and regulatory networks of the LEA protein family have not been extensively studied in conifers or gymnosperms. Pinus tabuliformis, a conifer with high ecological and economic values and with high-quality genome sequence, is an ideal candidate for such studies. Here, a total of 104 LEA genes were identified from P. tabuliformis, and we renamed them according to their subfamily group: PtLEA1-PtLEA92 (group LEA1-LEA6), PtSMP1-PtSMP6 (group seed maturation protein) and PtDHN1-PtDHN6 (group Dehydrin). While the sequence structure of P. tabuliformis LEA genes are conserved, their physicochemical properties exhibit unique characteristics within different subfamily groupings. Notably, the abundance of low-temperature responsive elements in PtLEA genes was observed. Using annual rhythm and temperature gradient transcriptome data, PtLEA22 was identified as a key gene that responds to low-temperature induction while conforming to the annual cycle of cold acclimation. Overexpression of PtLEA22 enhanced Arabidopsis freezing tolerance. Furthermore, several transcription factors potentially co-expressed with PtLEA22 were validated using yeast one-hybrid and dual-luciferase assays, revealing that PtDREB1 could directly bind PtLEA22 promoter to positively regulate its expression. These findings reveal the genome-wide characterization of P. tabuliformis LEA genes and their importance in the cold acclimation, while providing a theoretical basis for studying the molecular mechanisms of cold acclimation in conifers.

Analysis of Cyperus esculentus SMP family genes reveals lineage-specific evolution and seed desiccation-like transcript accumulation during tuber maturation

Seed maturation proteins (SMPs) represent a class of late embryogenesis-abundant proteins that are highly hydrophilic and abundant in mature seeds. Although it has been proven that SMPs are indispensable for the desiccation resistance of orthodox seeds , information on their putative roles in the desiccation resistance of non-seed storage organs is still scarce. This study presents a first transcriptome-based characterization of SMP family genes in tigernut ( Cyperus esculentus L., Cyperaceae), whose underground tubers represent a unique system that contains a considerably low moisture content of less than 10% without affecting the sprouting capability. Five family members identified from the full-length tigernut transcriptome are relatively more than four found in the genome of Carex littledalei , another Cyperaceae plant. Moreover, 69 family genes were also identified from 15 representative plant species and comparative genomics analysis supports the monogenic origin of this special family as well as lineage-specific evolution after monocot-eudicot split. In the Cyperaceae lineage, four clades present in the last common ancestor of tigernut and C. littledalei , which represent four out of 11 orthogroups proposed in this study, are more likely to result from transposed and dispersed duplications. In contrast to seed- or embryo-preferential expression of SMP genes in seed-bearing plants (e.g. rice and maize), transcriptional profiling and qRT-PCR analysis showed that most CeSMP genes are predominantly expressed in late stages of developmental tigernut tubers, negatively correlating with the moisture content. This seed desiccation-like accumulation of CeSMP transcripts during tuber maturation implies their putatively important roles in the desiccation resistance of tigernut tubers as observed in orthodox seeds. These findings not only improve the knowledge on lineage-specific evolution of the SMP gene family, but also provide valuable information for further functional analysis and genetic improvement in tigernut. • This is a first transcriptome-based analysis of SMP family genes in tigernut. • Comparative genomics analysis reveals the monogenic origin of the SMP family. • Lineage-specific family evolution was observed after monocot-eudicot split. • CeSMP transcripts are negatively correlating the moisture content of tubers. • Accumulation of CeSMPs in mature tubers implies a role in desiccation resistance.

Genome-wide identification and expression analyses of late embryogenesis abundant (LEA) gene family in tobacco (Nicotiana tabacum L.) reveal their function in abiotic stress responses

Late embryogenesis abundant (LEA) proteins play an important role in plant growth and response to abiotic stresses. However the late embryogenesis abundant (LEA) gene family in Nicotiana tabacum has not been systematically studied. In this study, 123 NtLEA genes were identified in Nicotiana tabacum, and divided into 8 groups, including LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, DHN (dehydratin) and SMP (Seed Maturation Protein). The LEA_2 group is the most abundant of the NtLEA family. The gene structure, conserved motifs, subcellular localization and physicochemical properties of the NtLEA genes were analyzed. RNA-seq and qPCR analyses showed that the NtLEA genes were significantly induced under two different abiotic stresses and showed different expression patterns. The expression patterns of 35 NtLEA genes responding to ABA and 3 NtLEA genes responding to NaCl abiotic stress, respectively, were characterized. The protein–protein interaction network revealed that most NtLEA proteins (>78%) had the potential function to enhance tobacco resistance to abiotic stress. The transcriptional regulatory network showed that 21 transcription factor families were involved in regulating the expression of the NtLEA genes. These results are beneficial for future studies of the function of the NtLEA genes.

Stage-specific protein regulation during somatic embryo development of Carica papaya L. ‘Golden’

Somatic embryogenesis is an important biotechnological technique for large-scale propagation of elite genotypes. Identifying stage-specific compounds associated with somatic embryo development can help elucidate the ontogenesis of Carica papaya L. somatic embryos and improve tissue culture protocols. To identify the stage-specific proteins that are present during the differentiation of C. papaya somatic embryos, proteomic analyses of embryos at the globular, heart, torpedo and cotyledonary developmental stages were performed. Mass spectrometry data have been deposited in the ProteomeXchange with the dataset identifier PXD021107. Comparative proteomic analyses revealed a total of 801 proteins, with 392 classified as differentially accumulated proteins in at least one of the developmental stages. The globular-staged presented a higher number of unique proteins (16), and 7 were isoforms of 60S ribosomal proteins, suggesting high translational activity at the beginning of somatic embryogenesis. Proteins related to mitochondrial metabolism accumulated to a high degree at the early developmental stages and then decreased with increasing development, and they contributed to cell homeostasis in early somatic embryos. A progressive increase in the accumulation of vicilin, late embryogenesis abundant proteins and chloroplastic proteins that lead to somatic embryo maturation was also observed. The differential accumulation of acetylornithine deacetylase and S-adenosylmethionine synthase 2 proteins was correlated with increases in putrescine and spermidine contents, which suggests that both polyamines should be tested to determine whether they increase the conversion rates of globular- to cotyledonary-staged somatic embryos. Taken together, the results showed that somatic embryo development in C. papaya is regulated by the differential accumulation of proteins, with ribosomal and mitochondrial proteins more abundant during the early somatic embryo stages and seed maturation proteins more abundant during the late stages.

Liquid-liquid phase separation promotes animal desiccation tolerance

Proteinaceous liquid-liquid phase separation (LLPS) occurs when a polypeptide coalesces into a dense phase to form a liquid droplet (i.e., condensate) in aqueous solution. In vivo, functional protein-based condensates are often referred to as membraneless organelles (MLOs), which have roles in cellular processes ranging from stress responses to regulation of gene expression. Late embryogenesis abundant (LEA) proteins containing seed maturation protein domains (SMP; PF04927) have been linked to storage tolerance of orthodox seeds. The mechanism by which anhydrobiotic longevity is improved is unknown. Interestingly, the brine shrimp Artemia franciscana is the only animal known to express such a protein (AfrLEA6) in its anhydrobiotic embryos. Ectopic expression of AfrLEA6 (AWM11684) in insect cells improves their desiccation tolerance and a fraction of the protein is sequestered into MLOs, while aqueous AfrLEA6 raises the viscosity of the cytoplasm. LLPS of AfrLEA6 is driven by the SMP domain, while the size of formed MLOs is regulated by a domain predicted to engage in protein binding. AfrLEA6 condensates formed in vitro selectively incorporate target proteins based on their surface charge, while cytoplasmic MLOs formed in AfrLEA6-transfected insect cells behave like stress granules. We suggest that AfrLEA6 promotes desiccation tolerance by engaging in two distinct molecular mechanisms: by raising cytoplasmic viscosity at even modest levels of water loss to promote cell integrity during drying and by forming condensates that may act as protective compartments for desiccation-sensitive proteins. Identifying and understanding the molecular mechanisms that govern anhydrobiosis will lead to significant advancements in preserving biological samples.

Proteomic Analysis of Lipid Droplets in Sesamum indicum

We attempted to identify the total proteome in sesame lipid droplets. Results from two-dimensional electrophoresis showed 139 protein spots in lipid droplet samples. Each spot was isolated, digested with trypsin, and applied to liquid chromatography–tandem mass spectrometry (Q-Tof Premier). As a result, 103 spots were identified. Although oleosin, caleosin, and steroleosin are known major components of the lipid droplet, many other proteins were also found in the lipid droplet. In addition to the three major proteins, TAG factor protein, glyceraldehyde-3-phosphate dehydrogenase, F1 ATPase, 70-kDa heat shock protein, seed maturation protein PM24, and 11S globulin precursor isoforms 3 and 4 were found in the lipid droplet. Three types of oleosins, 15-, 15.5-, and 17-kDa were present in the sesame lipid droplet, and the 15.5-kDa oleosin had high homology with oleosin from Coffea canephora. It has been shown by acid phosphatase treatment that oleosin proteins contain phosphate groups. Protein disulfide-isomerase 2 precursor, calreticulin-1, and BiP, which are known as marker proteins of the endoplasmic reticulum, were found as the components of the lipid droplet. Immunoconfocal microscopy was used to show that 11S globulin precursor isoform 3 and 4 were indeed localized in the lipid droplet. The presence of 11S globulin in the lipid droplets suggested a new mechanism for the lipid droplet formation.

Membraneless Organelles and Viscosity Changes during Desiccation: A New Phase in Anhydrobiosis

AfrLEA6 is an intrinsically disordered protein found in the anhydrobiotic cysts of Artemia franciscana. AfrLEA6 shares homology with plant seed maturation proteins (SMPs), which have been linked to the longevity of orthodox seeds in the desiccated state. Therefore, it was hypothesized that ectopically expressing AfrLEA6 will increase the desiccation tolerance of a desiccation‐sensitive Drosophila melanogaster (Kc167) cell line. Surprisingly, ectopically expressed AfrLEA6 was found to undergo a liquid‐liquid phase separation (LLPS) in the cytoplasm of Kc167 cells. The overall architecture of the protein is composed of two SMP domains connected with a glycine‐ and proline‐rich spacer to a potential protein‐binding domain. It was determined that AfrLEA6 LLPS is dependent on the SMP domains, regulated by polysome assembly and disassembly, and may best be described as a specialized biphasic stress granule. It is speculated that AfrLEA6 stress granules protect desiccation‐sensitive proteins prior to glassy‐state formation during water loss. This hypothesis was strengthened by the observation that expression of AfrLEA6 in Kc167 cells increases their desiccation tolerance. Unexpectedly, AfrLEA6 also increases intracellular viscosity during desiccation compared to control cells, which reduced plasma membrane fusion between adjacent cells and may protect against internal sheer stresses. These data agree with recently reported cellular dielectrophoretic data, that AfrLEA6 expressed in Kc167 cells underwent a shift to become more entangled when cells were exposed to osmotic stress, thereby suggesting an increase intracellular viscosity. In A. franciscana, a rapid increase in cytoplasmic viscosity at relatively high‐water contents may be key to successfully entering the anhydrobiotic state, while trehalose glass formation at relatively low‐water contents may create a more stable intracellular environment for long‐term dormancy.Support or Funding InformationSupported by NSF IOS‐1659970.

Identification of a seed maturation protein gene from Coffea arabica (CaSMP) and analysis of its promoter activity in tomato.

A seed maturation protein gene (CaSMP) from Coffea arabica is expressed in the endosperm of yellow/green fruits. The CaSMP promoter drives reporter expression in the seeds of immature tomato fruits. In this report, an expressed sequence tag-based approach was used to identify a seed-specific candidate gene for promoter isolation in Coffea arabica. The tissue-specific expression of the cognate gene (CaSMP), which encodes a yet uncharacterized coffee seed maturation protein, was validated by RT-qPCR. Additional expression analysis during coffee fruit development revealed higher levels of CaSMP transcript accumulation in the yellow/green phenological stage. Moreover, CaSMP was preferentially expressed in the endosperm and was down-regulated during water imbibition of the seeds. The presence of regulatory cis-elements known to be involved in seed- and endosperm-specific expression was observed in the CaSMP 5'-upstream region amplified by genome walking (GW). Additional histochemical analysis of transgenic tomato (cv. Micro-Tom) lines harboring the GW-amplified fragment (~ 1.4kb) fused to uidA reporter gene confirmed promoter activity in the ovule of immature tomato fruits, while no activity was observed in the seeds of ripening fruits and in the other organs/tissues examined. These results indicate that the CaSMP promoter can be used to drive transgene expression in coffee beans and tomato seeds, thus representing a promising biotechnological tool.

A Polypeptide from the Brine Shrimp Artemia Franciscana is Related to Plant Seed Maturation Proteins and Protects Lactate Dehydrogenase During Freezing and Desiccation

Several anhydrobiotic organisms accumulate hydrophilic polypeptides, including Late Embryogenesis Abundant (LEA) proteins, before the onset of dehydration. In the brine shrimp A. franciscana, multiple LEA proteins are expressed in the desiccation tolerant life‐history stage, and these proteins localize to different subcellular compartments. The protein AfrLEA6 contains a seed maturation protein (SMP) superfamily domain (pfam04927) and shows homologies to SMP proteins from plants (e.g. Nicotiana sylvestris, XP_009764712.1; e‐value 2e‐04). Even though the expression of LEA proteins is closely related with desiccation tolerance, the precise function and purpose of AfrLEA6 in A. franciscana is unknown. To better understand the role of LEA proteins in desiccation tolerance, large quantities of the protein are needed for functional studies. Therefore, an Escherichia coli expression system was chosen to generate sufficient quantities of AfrLEA6 for in vitro studies. Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) mediated induction using the vector pTXB1 in BL21 cells (E. coli) yielded approximately 20 mg of AfrLEA6 protein per liter of culture. Intein mediated purification with a chitin‐binding tag allowed for a single affinity chromatography step and avoids the use of additional amino acids that remain on the protein of interest. This system yielded AfrLEA6 at 95% purity after optimization. However, the c‐terminal amino acids penultimate to the beginning of the intein protein played a crucial role in the recovery efficiency using dithiothreitol (DTT) to induce intein cleavage. Maintaining the c‐terminal aspartic acid (Asp) residue found in the native AfrLEA6 protein yielded almost no cleavage when treated with DTT. However, when Asp was mutated to glutamic acid or alanine, cleavage efficiency was greatly improved. To evaluate potential protective properties of AfrLEA6 against freezing, heat‐denaturation, or desiccation induced denaturation, lactate dehydrogenase was chosen as model enzyme. In presence of 25μg ml−1 AfrLEA6 recovery of LDH activity after desiccation was significantly higher compared to control or in presence of lysozyme, BSA or the group 1 LEA protein AfrLEA1.1. In absence of AfrLEA6 and presence of lysozyme or BSA only about 25%‐50% of LDH activity was recovered, while at a AfrLEA6‐to‐LDH mass ratio of 5:1 nearly 70% of enzyme activity was protected. Similarly, 82% recovery of LDH activity was found after liquid nitrogen freezing at a AfrLEA6‐to‐LDH mass ratio of 10:1 while in presence of LEA1.1, BSA, or and lysozyme 64%, 59%, or 40% was recovered. Recovery of LDH activity after heating to 46°C, was also significantly improved in presence of AfrLEA6 compared to the other proteins, but the enzyme lost most of its activity within 30 minutes of temperature challenge under all conditions. Further research to elucidate the mechanism by which AfrLEA6 protects enzymes from denaturation during freezing, drying, and elevated temperature may offer novel avenues for biotechnological applications ranging from cell stabilization to storage of biomedical relevant proteins.Support or Funding InformationThis project was supported by the National Sciences Foundation (IOS‐1659970/IOS‐1457061).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteomic and metabolomic analysis of minimax and Williams 82 soybeans grown under two different conditions

MiniMax was developed as a model soybean for research because of its small stature, limited planting space, and rapid generation time. In the present study, the proteins, isoflavones, and fatty acid methyl esters (FAMEs) were compared between dwarf (MiniMax) and commercially cultivated (Williams 82) soybean genotypes under two growing conditions (greenhouse and field). The protein profile of the soybean extracts by 2D-PAGE showed at least 16 of 716 spots that were differently expressed in MiniMax and Williams 82. These spots were primarily six proteins namely, seed biotin, β-conglycinin, glycinin, seed-maturation protein, glutathione-S-transferase, and sucrose binding protein. In both genotypes, higher content of isoflavone aglycones was observed in greenhouse grown samples as compared to the field grown samples. However, only field grown Willams 82 had higher levels of FAMEs as compared to other three groups. This study shows that both growing conditions and cultivars significantly influence the compositional content of metabolites. Practical applications The global demand of soybean and its products has increased due to its high protein and isoflavones content. Researchers are actively developing new genotypes with improved yields, greater drought and disease tolerance, and reduced growth time. MiniMax, a dwarf genotype of soybean showed early flowering, seed setting, and maturation traits due to its multiple dwarfing genes. This characteristic feature enables researchers to study effects of various treatments on soybean in a shorter period with multiple replications in a single growing season.

Genome-wide identification and analysis of late embryogenesis abundant (LEA) genes in Prunus mume

Late embryogenesis abundant (LEA) proteins play important roles in plant desiccation tolerance. In this study, 30 LEA genes were identified from Chinese plum (Prunus mume) through genome-wide analysis. The PmLEA genes are distributed on all Chinese plum chromosomes except chromosome 3. Twelve (40%) and five PmLEA genes are arranged in tandem and segmental duplications, respectively. The PmLEA genes could be divided into eight groups (LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, PvLEA18, dehydrin and seed maturation protein). Ten gene conversion events were observed and most of them (70%) were identified in dehydrin group. Most PmLEA genes were highly expressed in flower (22/30) and up-regulated by ABA treatment (19/30).

Global gene expression profiles in developing soybean seeds

The gene expression profiles in soybean (Glycine max L.) seeds at 4 stages of development, namely, pod, 2-mm bean, 5-mm bean, and full-size bean, were examined by DNA microarray analysis. The total genes of each sample were classified into 4 clusters based on stage of development. Gene expression was strictly controlled by seed size, which coincides with the development stage. First, stage specific gene expression was examined. Many transcription factors were expressed in pod, 2-mm bean and 5-mm bean. In contrast, storage proteins were mainly expressed in full-size bean. Next, we extracted the genes that are differentially expressed genes (DEGs) that were extracted using the Rank products method of the Bioconductor software package. These DEGs were sorted into 8 groups using the hclust function according to gene expression patterns. Three of the groups across which the expression levels progressively increased included 100 genes, while 3 groups across which the levels decreased contained 47 genes. Storage proteins, seed-maturation proteins, some protease inhibitors, and the allergen Gly m Bd 28K were classified into the former groups. Lipoxygenase (LOX) family members were present in both the groups, indicating the multi-functionality with different expression patterns.

Assessment of the natural variation of low abundant metabolic proteins in soybean seeds using proteomics

Using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, we investigated the distribution of the low abundant proteins that are involved in soybean seed development in four wild and 12 cultivated soybean genotypes. We found proteomic variation of low abundant proteins involved in soybean seed metabolism within and between groups. The major proteomic variation among these 16 genotypes was observed in the seed maturation proteins, which consisted of 4–6 protein spots. All genotypes showed 3 spots of sucrose-binding protein except one wild genotype which had 2 spots. Two protein spots were detected for a triacylglycerol (TAG) accumulation factor protein in 13 genotypes, while one spot was detected in one ancestral and one wild genotype, and no spot was detected in one of the wild genotypes. All genotypes showed 3 spots of alcohol dehydrogenase except one Asian landrace and one wild genotype that had only one spot. Minor proteomic variation was detected in 16 genotypes for an elongation factor (EF2) and formate dehydrogenase, with apparent absence of EF2 in one landrace and formate dehydrogenase in another landrace genotype. No differences in the proteomic distribution of dehydrin-like protein, seed biotin-containing protein, glyceraldehyde 3-phosphate dehydrogenase, and fructose bisphosphate aldolase were detected. This information will be useful in selecting genotypes for breeding programs, and forms a baseline to which variation in transgenic varieties can be compared.

Proteomic analysis of seed germination under salt stress in soybeans

Soybean (Glycine max (L.) Merrill) is a salt-sensitive crop, and its production is severely affected by saline soils. Therefore, the response of soybean seeds to salt stress during germination was investigated at both physiological and proteomic levels. The salt-tolerant cultivar Lee68 and salt-sensitive cultivar N2899 were exposed to 100 mmol/L NaCl until radicle protrusion from the seed coat. In both cultivars, the final germination percentage was not affected by salt, but the mean germination times of Lee68 and N2899 were delayed by 0.3 and 1.0 d, respectively, compared with controls. In response to salt stress, the abscisic acid content increased, and gibberellic acid (GA₁+₃) and isopentenyladenosine decreased. Indole-3-acetic acid increased in Lee68, but remained unchanged in N2899. The proteins extracted from germinated seeds were separated using two-dimensional gel electrophoresis (2-DE), followed by Coomassie brilliant blue G-250 staining. About 350 protein spots from 2-DE gels of pH range 3 to 10 and 650 spots from gels of pH range 4 to 7 were reproducibly resolved, of which 18 protein spots showed changes in abundance as a result of salt stress in both cultivars. After matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of the differentially expressed proteins, the peptide mass fingerprint was searched against the soybean UniGene database and nine proteins were successfully identified. Ferritin and 20S proteasome subunit β-6 were up-regulated in both cultivars. Glyceraldehyde 3-phosphate dehydrogenase, glutathione S-transferase (GST) 9, GST 10, and seed maturation protein PM36 were down-regulated in Lee68 by salt, but still remained at a certain level. However, these proteins were present in lower levels in control N2899 and were up-regulated under salt stress. The results indicate that these proteins might have important roles in defense mechanisms against salt stress during soybean seed germination.

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

Expression of the Stress-Related Genes for Glutathione S-Transferase and Ascorbate Peroxidase in the Most-Glycinin-Deficient Soybean Cultivar Tousan205 during Seed Maturation

Global analysis of gene expression profiles in most-glycinin-deficient cultivar Tousan205, was performed by DNA microarray analysis. It was confirmed that Tousan205 lacks mRNA expression of three glycinin subunit precursor genes, G1 (A1aB1x), G2 (A2B1a), and G5 (A3B4), and lacks G4 (A5A4B3) protein. Most glycinin subunits were deficient in mature seeds of Tousan205. We compared the gene expression of Tousan205 with those of parent cultivar, Tamahomare, which was used for crossbreeding of Tousan205. As a result, Tousan205 exhibited higher expression of some seed maturation proteins, and stress-related genes such as glutathione S-transferase and ascorbate peroxidase. This result indicates the possibility that the decrease of main storage protein, glycinin causes stress in soybean.

Involvement of an R2R3-MYB transcription factor gene AtMYB118 in embryogenesis in Arabidopsis

MYB transcription factors play important roles in various developmental processes in plants. Here we report the characterization of AtMYB118, a gene encoding a putative R2R3-type MYB transcription factor, which expresses predominantly in siliques. Real-time quantitative PCR analysis and in situ hybridization showed that the transcripts of AtMYB118 were mainly detected in developing embryos. Constitutive over-expression of AtMYB118 resulted in pleiotropic phenotypes, including dwarfism, compact rosettes, backward curly-leaves, smaller flowers and siliques, and premature seed dehydration at the tip of siliques. Microarray analysis showed that many genes encoding proteins accumulated during embryogenesis were remarkably up-regulated in AtMYB118-over-expressed transgenic plants, including late embryogenesis abundant proteins (LEA proteins), storage proteins, seed maturation proteins, and proteins related with seed dehydration, desiccation and ABA signaling pathway. These results suggest that AtMYB118 may play an important role during embryogenesis and seed maturation.

The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought.

A New Branch of Endoplasmic Reticulum Stress Signaling and the Osmotic Signal Converge on Plant-specific Asparagine-rich Proteins to Promote Cell Death

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.