Satish C. Bhatla

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications.

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins-oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.

Salt stress-induced seedling growth inhibition coincides with differential distribution of serotonin and melatonin in sunflower seedling roots and cotyledons.

Indoleamines regulate a variety of physiological functions during the growth, morphogenesis and stress-induced responses in plants. Present investigations report the effect of NaCl stress on endogenous serotonin and melatonin accumulation and their differential spatial distribution in sunflower (Helianthus annuus) seedling roots and cotyledons using HPLC and immunohistochemical techniques, respectively. Exogenous serotonin and melatonin treatments lead to variable effect on hypocotyl elongation and root growth under NaCl stress. NaCl stress for 48 h increases endogenous serotonin and melatonin content in roots and cotyledons, thus indicating their involvement in salt-induced long distance signaling from roots to cotyledons. Salt stress-induced accumulation of serotonin and melatonin exhibits differential distribution in the vascular bundles and cortex in the differentiating zones of the primary roots, suggesting their compartmentalization in the growing region of roots. Serotonin and melatonin accumulation in oil body rich cells of salt-treated seedling cotyledons correlates with longer retention of oil bodies in the cotyledons. Present investigations indicate the possible role of serotonin and melatonin in regulating root growth during salt stress in sunflower. Effect of exogenous serotonin and melatonin treatments (15 μM) on sunflower seedlings grown in the absence or presence of 120 mM NaCl substantiates their role on seedling growth. Auxin and serotonin biosynthesis are coupled to the common precursor tryptophan. Salt stress-induced root growth inhibition, thus pertains to partial impairment of auxin functions caused by increased serotonin biosynthesis. In seedling cotyledons, NaCl stress modulates the activity of N-acetylserotonin O-methyltransferase (HIOMT; EC 2.1.1.4), the enzyme responsible for melatonin biosynthesis from N-acetylserotonin.

Sodium chloride stress induces nitric oxide accumulation in root tips and oil body surface accompanying slower oleosin degradation in sunflower seedlings

Present work highlights the involvement of endogenous nitric oxide (NO) in sodium chloride (NaCl)-induced biochemical regulation of seedling growth in sunflower (Helianthus annuus L., cv. Morden). The growth response is dependent on NaCl concentration to which seedlings are exposed, they being tolerant to 40 mM NaCl and showing a reduction in extension growth at 120 mM NaCl. NaCl sensitivity of sunflower seedlings accompanies a fourfold increase in Na(+) /K(+) ratio in roots (as compared to that in cotyledons) and rapid transport of Na(+) to the cotyledons, thereby enhancing Na(+) /K(+) ratio in cotyledons as well. A transient increase in endogenous NO content, primarily contributed by putative NOS activity in roots of 4-day-old seedlings subjected to NaCl stress and the relative reduction in Na(+) /K(+) ratio after 4 days, indicates that NO regulates Na(+) accumulation, probably by affecting the associated transporter proteins. Root tips exhibit an early and transient enhanced expression of 4,5-diaminofluorescein diacetate (DAF-2DA) positive NO signal in the presence of 120 mM NaCl. Oil bodies from 2-day-old seedling cotyledons exhibit enhanced localization of NO signal in response to 120 mM NaCl treatment, coinciding with a greater retention of the principal oil body membrane proteins, i.e. oleosins. Abolition of DAF positive fluorescence by the application of specific NO scavenger [2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO)] authenticates the presence of endogenous NO. These novel findings provide evidence for a possible protective role of NO during proteolytic degradation of oleosins prior to/accompanying lipolysis.

Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25–80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.

Mechanisms of nitric oxide crosstalk with reactive oxygen species scavenging enzymes during abiotic stress tolerance in plants

ABSTRACT Nitric oxide (NO) acts in a concentration and redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging reactive oxygen species (ROS), such as superoxide anions (O2−*), to form peroxynitrite (ONOO−) or by acting as a signaling molecule, thereby altering gene expression. NO can interact with different metal centres in proteins, such as heme-iron, zinc–sulfur clusters, iron–sulfur clusters, and copper, resulting in the formation of a stable metal–nitrosyl complex or production of varied biochemical signals, which ultimately leads to modification of protein structure/function. The thiols (ferrous iron–thiol complex and nitrosothiols) are also involved in the metabolism and mobilization of NO. Thiols bind to NO and transport it to the site of action whereas nitrosothiols release NO after intercellular diffusion and uptake into the target cells. S-nitrosoglutathione (GSNO) also has the ability to transnitrosylate proteins. It is an NO˙ reservoir and a long-distance signaling molecule. Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins. The exact molecular mechanism(s) by which exogenous and endogenously generated NO (or reactive nitrogen species) modulate the induction of various genes affecting redox homeostasis, are being extensively investigated currently by various research groups. Present review provides an in-depth analysis of the mechanisms by which NO interacts with and modulates the activity of various ROS scavenging enzymes, particularly accompanying ROS generation in plants in response to varied abiotic stress.

Melatonin and nitric oxide regulate sunflower seedling growth under salt stress accompanying differential expression of Cu/Zn SOD and Mn SOD

Abstract Salinity results in significant reduction in sunflower (Helianthus annuus L.) seedling growth and excessive generation of reactive oxygen species (ROS). Present work highlights the possible role of melatonin as an antioxidant through its interaction with nitric oxide (NO), and as an early and long distance NaCl‐stress sensing signaling molecule in seedling cotyledons. Exogenous melatonin (15 &mgr;M)±NaCl (120 mM) inhibit seedling growth, which is also correlated with NO availability, accumulation of potential superoxide anion (O2•‐) and peroxynitrite anion (ONOO‐), extent of tyrosine‐nitration of proteins, spatial localization and activity of superoxide dismutase (SOD) isoforms. NO acts as a positive modulator of melatonin accumulation in seedling cotyledons as a long‐distance signaling response. Modulation of superoxide anion and peroxynitrite anion content by melatonin highlights its crucial role in combating deleterious effects of ROS and reactive nitrogen species (RNS). Present findings provide evidence for an interaction between melatonin and NO in their effect on seedling growth under salt stress accompanying differential modulation of two SOD isoforms, i.e. Cu/Zn SOD and Mn SOD. HighlightsProbable melatonin‐NO interaction in redox homeostasis maintenance.Melatonin as an early and long distance signaling molecule in sunflower seedlings.Differential modulation of SOD isoforms by melatonin in seedlings under salt stress.

Recent developments in the localization of oil body-associated signaling molecules during lipolysis in oilseeds

Prior to and/or accompanying lipolytic degradation of triacylglycerols (TAGs) during seed germination in oilseeds, certain enzymatic and non-enzymatic signaling molecules are expressed on the oil body membranes. These include certain proteases, lipoxygenase, phospholipase A2 and lipase. Although enough biochemical investigations have demonstrated their activities, recent developments in the in situ localization of these signaling molecules in germinating oilseeds, have enhanced our understanding in this field. This is evident from the temporal and spatial changes observed in the expression pattern of some of these molecules. Present review aims at providing an up-to-date account of these recent developments in the author’s and other laboratories, which are largely based on fluorescence microscopic and confocal laser scanning microscopic (CLSM) imaging of the molecular changes using specific fluorescent probes. A model for the molecular events associated with oil body mobilization is also being presented.

Light-enhanced oil body mobilization in sunflower seedlings accompanies faster protease action on oleosins

Abstract Germination of sunflower ( Helianthus annuus L.) seeds in light is accompanied by greater susceptibility of oil bodies for lipolytic action following enhanced oleosin mobilization than in the dark. The 16- and 17.5-kDa oleosins are mobilized within the first 4 d of seedling growth in light, whereas 20-kDa oleosin remains detectable. Oleosin mobilization is slower in the dark and all three oleosins remain detectable until 7 d of seedling growth. Light-grown seedlings show higher activity of fatty acyl-ester hydrolase (EC 3.1.1.1) mainly due to greater expression of its major (40–50 kDa) isoforms. Increased susceptibility of oil bodies to lipolytic action in light-grown seedlings shows a correlation with higher activity of a cytosolic 65-kDa protease, oleosin mobilization and relative accumulation of 11-kDa protease-protected fragment. These observations support the view that the expression of 65-kDa protease is enhanced in light and it could be considered as a component of light-enhanced lipolysis.

Signaling role of phospholipid hydroperoxide glutathione peroxidase (PHGPX) accompanying sensing of NaCl stress in etiolated sunflower seedling cotyledons

Sunflower seedlings subjected to 120 mM NaCl stress exhibit high total peroxidase activity, differential expression of its isoforms and accumulation of lipid hydroperoxides. This coincides with high specific activity of phospholipid hydroperoxide glutathione peroxidase (PHGPX) in the 10,000g supernatant from the homogenates of 2–6 d old seedling cotyledons. An upregulation of PHGPX activity by NaCl is evident from Western blot analysis. Confocal laser scanning microscopic (CLSM) analysis of sections of cotyledons incubated with anti-GPX4 (PHGPX) antibody highlights an enhanced cytosolic accumulation of PHGPX, particularly around the secretory canals. Present work, thus, highlights sensing of NaCl stress in sunflower seedlings in relation with lipid hydroperoxide accumulation and its scavenging through an upregulation of PHGPX activity in the cotyledons.

Nitric oxide modulates specific steps of auxin-induced adventitious rooting in sunflower

Present work on indole-3-acetic acid (IAA)-induced adventitious rooting in sunflower hypocotyl highlights a clear demarcation of nitric oxide (NO)-dependent and NO-independent roles of auxin in this developmental process. Of the three phases of adventitious rooting, induction is strictly auxin-dependent though initiation and extension are regulated by an interaction of IAA with NO. A vital role of auxin-efflux transporters (PIN) is also evident from 1-napthylphthalamic acid (NPA)-triggered suppression of adventitious roots (AR). Use of actin depolymerizing agent, Latrunculin B (Lat B), has demonstrated the necessity of functional actin filaments in auxin-induced AR response, possibly through its effect on actin-mediated recycling of auxin transporter proteins. Thus, evidence for a linkage between IAA, NO and actin during AR formation has been established.