Yizhi Zheng

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half; PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.

Pongamia (Millettia pinnata syn. Pongamia pinnata), an oilseed legume species, is emerging as potential feedstock for sustainable biodiesel production. Breeding Pongamia for favorable traits in commercial application will rely on a comprehensive understanding of molecular mechanism regulating oil accumulation during its seed development. To date, only limited genomic or transcript sequences are available for Pongamia, while a temporal transcriptome profiling of developing seeds is still lacking in this species. In this work, we conducted a time-series analysis of morphological and physiological characters, oil contents and compositions, as well as global gene expression profiles in developing Pongamia seeds. Firstly, three major developmental phases were characterized based on the combined evidences from embryonic shape, seed weight, seed moisture content, and seed color. Then, the gene expression levels at these three phases were quantified by RNA-Seq analyses with three biological replicates from each phase. Nearly 94% of unigenes were expressed at all three phases, whereas only less than 2% of unigenes were exclusively expressed at one of these phases. A total of 8881 differentially expressed genes (DEGs) were identified between phases. Furthermore, the qRT-PCR analyses for 10 DEGs involved in lipid metabolism demonstrated a good reliability of our RNA-Seq data in temporal gene expression profiling. We observed a dramatic increase in seed oil content from the embryogenesis phase to the early seed-filling phase, followed by a steady and moderate increase towards the maximum at the desiccation phase. We proposed that a highly active expression of most genes related to fatty acid (FA) and triacylglycerol (TAG) biosynthesis at the embryogenesis phase might trigger both the substantial oil accumulation and the membrane lipid synthesis for rapid cell proliferation at this phase, while a concerted reactivation of TAG synthesis-related genes at the desiccation phase might further promote storage lipid synthesis to achieve the maximum content of seed oils. This study not only built a bridge between gene expression profiles and oil accumulation in developing seeds, but also laid a foundation for future attempts on genetic engineering of Pongamia varieties to acquire higher oil yield or improved oil properties for biofuel applications.

Intrinsically disordered proteins (IDPs) play a variety of important physiological roles in all living organisms. However, there is no comprehensive analysis of the abundance of IDPs associated with environmental stress in plants. Here, we show that a set of heat-stable proteins (i.e., proteins that do not denature after boiling at 100 °C for 10 min) was present in R_0mm and R_15mm radicles (i.e., before radicle emergence and 15 mm long radicles) of soybean (Glycine max) seeds. This set of 795 iTRAQ-quantified heat-stable proteins contained a high proportion of wholly or highly disordered proteins (15%), which was significantly higher than that estimated for the whole soybean proteome containing 55,787 proteins (9%). The heat-stable proteome of soybean radicles that contain many IDPs could protect lactate dehydrogenase (LDH) during freeze–thaw cycles. Comparison of the 795 heat-stable proteins in the R_0mm and R_15mm soybean radicles revealed that many of these proteins changed abundance during seedling growth with 170 and 89 proteins being more abundant in R_0mm and R_15mm, respectively. KEGG analysis identified 18 proteins from the cysteine and methionine metabolism pathways and nine proteins from the phenylpropanoid biosynthesis pathway. As an important type of IDP related to stress, 30 late embryogenesis abundant proteins were also found. Ten selected proteins with high levels of predicted intrinsic disorder were able to efficiently protect LDH from the freeze–thaw-induced inactivation, but the protective ability was not correlated with the disorder content of these proteins. These observations suggest that protection of the enzymes and other proteins in a stressed cell can be one of the biological functions of plant IDPs.

Late embryogenesis abundant (LEA) proteins are widely distributed among plant species, where they contribute to abiotic stress tolerance. LEA proteins can be classified into seven groups according to conserved sequence motifs. The PM1 protein from soybean, which belongs to the Pfam LEA_1 group, has been shown previously to be at least partially natively unfolded, to bind metal ions and potentially to stabilize proteins and membranes. Here, we investigated the role of the PM1 C-terminal domain and in particular the multiple histidine residues in this half of the protein. We constructed recombinant plasmids expressing full-length PM1 and two truncated forms, PM1-N and PM1-C, which represent the N- and C-terminal halves of the protein, respectively. Immunoblotting and cross-linking experiments showed that full-length PM1 forms oligomers and high molecular weight (HMW) complexes in vitro and in vivo, while PM1-C, but not PM1-N, also formed oligomers and HMW complexes in vitro. When the histidine residues in PM1 and PM1-C were chemically modified, oligomerization was abolished, suggesting that histidines play a key role in this process. Furthermore, we demonstrated that high Cu²⁺ concentrations promote oligomerization and induce PM1 and PM1-C to form HMW complexes. Therefore, we speculate that PM1 proteins not only maintain ion homeostasis in the cytoplasm, but also potentially stabilize and protect other proteins during abiotic stress by forming a large, oligomeric molecular shield around biological targets.