Beneficial implications of sugar beet proteinase inhibitor BvSTI on plant architecture and salt stress tolerance in Lotus corniculatus L.

Food demands of increasing human population dictate intensification of livestock production, however, environmental stresses could jeopardize producers' efforts. Forage legumes suffer from yield losses and poor nutritional status due to salinity increase of agricultural soils. As tools aimed to reduce negative impacts of biotic or abiotic stresses, proteinase inhibitors (PIs) have been promoted for biotechnological improvements. In order to increase tolerance of Lotus corniculatus L. to salt stress, serine PI, BvSTI, was introduced into this legume using Agrobacterium rhizogenes, with final transformation efficiency of 4.57%. PCR, DNA gel-blot, RT-PCR and in-gel protein activity assays confirmed the presence and activity of BvSTI products in transformed lines. Plants from three selected transgenic lines (21, 73 and 109) showed significant alterations in overall phenotypic appearance, corresponding to differences in BvSTI accumulation. Lines 73 and 109 showed up to 7.3-fold higher number of tillers and massive, up to 5.8-fold heavier roots than in nontransformed controls (NTC). Line 21 was phenotypically similar to NTC, accumulated less BvSTI transcripts and did not exhibit an additional band of recombinant trypsin inhibitor as seen in lines 73 and 109. Exposure of the transgenic lines to NaCl revealed different levels of salt stress susceptibility. The NaCl sensitivity index, based on morphological appearance and chlorophyll concentrations showed that lines 73 and 109 were significantly less affected by salinity than NTC or line 21. High level of BvSTI altered morphology and delayed salt stress related senescence, implicating BvSTI gene as a promising tool for salinity tolerance improvement trials in L. corniculatus.

Hairy root culture as a valuable tool for allelopathic studies in apple.

Allelopathic plants exploit their chemical 'weapons' to prevail over the competition, suppress neighboring plants and consequently use the available resources more efficiently. However, the investigation of plant allelopathic interactions in rhizosphere is difficult to perform because of its high complexity due to interactions of biotic and abiotic factors. Thus, autonomous, aseptic root cultures of apple (Malus × domestica Borkh.) could facilitate allelopathic studies. We report on the successful genetic transformation of apple cultivars Melrose, Golden Delicious, Cadel and Gloster using Agrobacterium rhizogenes (Riker et al. 1930) Conn 1942 strain 15834 and for the first time the establishment of apple autonomous and permanent in vitro hairy root cultures that could be used as a new tool for apple allelopathic assays. Molecular characterization of transgenic hairy root lines was conducted to elucidate the possible relationship between expression of T-DNA genes and root growth characteristics that include branching. Similar content of phenolic acids (chlorogenic, caffeic, syringic, p-coumaric and ferulic), glycosilated flavonoids (rutin, quercitrin, isoquercitrin, kaempferol-3-glucoside) and flavonoid aglycones (quercetin and naringenin), and dihydrochalcone phloridzin, was detected in untransformed and transgenic apple root tissue by ultra high-performance liquid chromatography with mass spectrometry (UHPLC/(+/-)HESI-MS/MS) analyses, confirming that genetic transformation did not disturb secondary metabolite production in apple. Chlorogenic and caffeic acids and dihydrochalcones phloridzin and phloretin were detected as putative allelochemicals exuded into the growth medium in which transgenic roots were maintained for 4 weeks. Apple hairy root exudates significantly affected shoot and root development and growth of test plant Arabidopsis thaliana (L.) Heynh. seedlings after 5 or 10 days of treatment. Additionally, core cell-cycle genes CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 were down regulated in Arabidopsis shoots suggesting, in part, their role in inhibition of shoot growth. The present work highlighted an autonomous and permanent in vitro hairy root culture system as a valuable tool for studying allelopathic potential of apple, offering new perspective for allelopathy background elucidation in this important fruit species.

Transcriptome analysis of sugar beet root maggot (Tetanops myopaeformis) genes modulated by the Beta vulgaris host.

Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40% of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of 18 selected EST clones were confirmed by RT-PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed.

Co-expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development.

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Dragacevka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long-term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.

Extraordinary Adaptive Plasticity of Colorado Potato Beetle: "Ten-Striped Spearman" in the Era of Biotechnological Warfare.

Expanding from remote areas of Mexico to a worldwide scale, the ten-striped insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), has risen from being an innocuous beetle to a prominent global pest. A diverse life cycle, phenotypic plasticity, adaptation to adverse conditions, and capability to detoxify or tolerate toxins make this insect appear to be virtually "indestructible". With increasing advances in molecular biology, tools of biotechnological warfare were deployed to combat CPB. In the last three decades, genetically modified potato has created a new challenge for the beetle. After reviewing hundreds of scientific papers dealing with CPB control, it became clear that even biotechnological means of control, if used alone, would not defeat the Colorado potato beetle. This control measure once again appears to be provoking the potato beetle to exhibit its remarkable adaptability. Nonetheless, the potential for adaptation to these techniques has increased our knowledge of this pest and thus opened possibilities for devising more sustainable CPB management programs.

Growth and development of Colorado potato beetle larvae, Leptinotarsa decemlineata, on potato plants expressing the oryzacystatin II proteinase inhibitor.

Plant proteinase inhibitors (PIs) are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. PI oryzacystatin II (OCII), isolated from rice, showed potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate the applicability of the OCII gene in enhancing plant defence, OCII-transformed potatoes were bioassayed for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say). Feeding on transformed leaves of potato cultivars Desiree and Jelica significantly affected larval growth and development, but did not change mortality rates. During the L2 and L3 developmental stages larvae consumed the OCII-transformed foliage faster as compared to the nontransformed control. Also these larvae reached the prepupal stage (end of L4 stage) 2 days earlier than those fed on control leaves. However, the total amounts of consumed OCII-transformed leaves were up to 23% lower than of control, and the maximal weights of prepupal larvae were reduced by up to 18% as compared to larvae fed on nontransformed leaves. The reduction in insect fitness reported in this study in combination with other control measures, could lead to improved CPB resistance management in potato.

Hormonal and metabolic regulation of tomato fruit sink activity and yield under salinity.

Salinization of water and soil has a negative impact on tomato (Solanum lycopersicum L.) productivity by reducing growth of sink organs and by inducing senescence in source leaves. It has been hypothesized that yield stability implies the maintenance or increase of sink activity in the reproductive structures, thus contributing to the transport of assimilates from the source leaves through changes in sucrolytic enzymes and their regulation by phytohormones. In this study, classical and functional physiological approaches have been integrated to study the influence of metabolic and hormonal factors on tomato fruit sink activity, growth, and yield: (i) exogenous hormones were applied to plants, and (ii) transgenic plants overexpressing the cell wall invertase (cwInv) gene CIN1 in the fruits and de novo cytokinin (CK) biosynthesis gene IPT in the roots were constructed. Although salinity reduces fruit growth, sink activity, and trans-zeatin (tZ) concentrations, it increases the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) during the actively growing period (25 days after anthesis). Indeed, exogenous application of the CK analogue kinetin to salinized actively growing fruits recovered sucrolytic activities (mainly cwInv and sucrose synthase), sink strength, and fruit weight, whereas the ethylene-releasing compound ethephon had a negative effect in equivalent non-stressed fruits. Fruit yield was increased by both the constitutive expression of CIN1 in the fruits (up to 4-fold) or IPT in the root (up to 30%), owing to an increase in the fruit number (lower flower abortion) and in fruit weight. This is possibly related to a recovery of sink activity in reproductive tissues due to both (i) increase in sucrolytic activities (cwInv, sucrose synthase, and vacuolar and cytoplasmic invertases) and tZ concentration, and (ii) a decrease in the ACC levels and the activity of the invertase inhibitor. This study provides new functional evidences about the role of metabolic and hormonal inter-regulation of local sink processes in controlling tomato fruit sink activity, growth, and yield under salinity.

Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

Root-targeted biotechnology to mediate hormonal signalling and improve crop stress tolerance.

Since plant root systems capture both water and nutrients essential for the formation of crop yield, there has been renewed biotechnological focus on root system improvement. Although water and nutrient uptake can be facilitated by membrane proteins known as aquaporins and nutrient transporters, respectively, there is a little evidence that root-localised overexpression of these proteins improves plant growth or stress tolerance. Recent work suggests that the major classes of phytohormones are involved not only in regulating aquaporin and nutrient transporter expression and activity, but also in sculpting root system architecture. Root-specific expression of plant and bacterial phytohormone-related genes, using either root-specific or root-inducible promoters or grafting non-transformed plants onto constitutive hormone producing rootstocks, has examined the role of root hormone production in mediating crop stress tolerance. Root-specific traits such as root system architecture, sensing of edaphic stress and root-to-shoot communication can be exploited to improve resource (water and nutrients) capture and plant development under resource-limited conditions. Thus, root system engineering provides new opportunities to maintain sustainable crop production under changing environmental conditions.

Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants.

Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100 mM NaCl for 22 d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K(+) (20%) and decreases in the toxic ion Na(+) (by 30%) and abscisic acid (by 20-40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75 mM NaCl for 90 d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield.

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.).

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a pI value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing patterns revealed the induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alfalfa tissues.

Insect feeding-induced differential expression of Beta vulgaris root genes and their regulation by defense-associated signals.

Root responses to insect pests are an area of plant defense research that lacks much information. We have identified more than 150 sugar beet root ESTs enriched for genes responding to sugar beet root maggot feeding from both moderately resistant, F1016, and susceptible, F1010, genotypes using suppressive subtractive hybridization. The largest number of identified F1016 genes grouped into the defense/stress response (28%) and secondary metabolism (10%) categories with a polyphenol oxidase gene, from F1016, identified most often from the subtractive libraries. The differential expression of the root ESTs was confirmed with RT-PCR. The ESTs were further characterized using macroarray-generated expression profiles from F1016 sugar beet roots following mechanical wounding and treatment of roots with the signaling molecules methyl jasmonate, salicylic acid and ethylene. Of the examined root ESTs, 20, 17 and 11% were regulated by methyl jasmonate, salicylic acid and ethylene, respectively, suggesting these signaling pathways are involved in sugar beet root defense responses to insects. Identification of these sugar beet root ESTs provides knowledge in the field of plant root defense and will lead to the development of novel control strategies for control of the sugar beet root maggot.

Biolistic transformation of highly regenerative sugar beet (Beta vulgaris L.) leaves.

Leaves of greenhouse-grown sugar beet (Beta vulgaris L.) plants that were first screened for high regeneration potential were transformed via particle bombardment with the uidA gene fused to the osmotin or proteinase inhibitor II gene promoter. Stably transformed calli were recovered as early as 7 weeks after bombardment and GUS-positive shoots regenerated 3 months after bombardment. The efficiency of transformation ranged from 0.9% to 3.7%, and stable integration of the uidA gene into the genome was confirmed by Southern blot analysis. The main advantages of direct bombardment of leaves to regenerate transformed sugar beet include (1) a readily available source of highly regenerative target tissue, (2) minimal tissue culture manipulation before and after bombardment, and (3) the overall rapid regeneration of transgenic shoots.

Pest and disease resistance enhanced by heterologous suppression of a Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2.

The functional role of the Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58 kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools.

Expression of Oryzacystatin I and II in Alfalfa Increases Resistance to the Root-Lesion Nematode.

ABSTRACT Digestive cysteine proteinases have been isolated from plant-parasitic nematodes as well as coleopteran and hemipteran insects. Phytocystatins, inhibitors of cysteine proteinases, are found in a number of plants where they may play a role in defense against pathogens and pests. The cDNAs of the phytocystatins from rice, oryzacystatin I (OC-I) and oryzacystatin II (OC-II), were expressed in alfalfa (Medicago sativa) plants under the control of the potato protease inhibitor II (PinII) promoter and the plants were evaluated for resistance to the root-lesion nematode (Pratylenchus penetrans). A PinII-beta-glucuronidase (GUS) gene was introduced into alfalfa to determine the pattern of gene expression from this promoter. Constitutive GUS expression was observed in leaf and root vascular tissue, and in some plants, expression was observed in leaf mesophyll cells. Mechanical wounding of leaves increased GUS expression approximately twofold over 24 h. Inoculation with root-lesion nematodes resulted in localized GUS expression. Populations of root-lesion nematodes in alfalfa roots from one line containing the PinII::OC-I transgene and one line containing the PinII::OC-II transgene were reduced 29 and 32%, respectively, compared with a transgenic control line. These results suggest that oryzacystatins have the potential to confer increased resistance to the root-lesion nematode in alfalfa.