OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced by cold and gibberellin in rice leaf sheath

Calcium-dependent protein kinases (CDPKs) play an important role in rice signal transduction, but the precise role of each individual CDPK is still largely unknown. Recently, a full-length cDNA encoding OsCDPK13 from rice seedling was isolated. To characterize the function of OsCDPK13, its responses to various stresses and hormones were analyzed in this study. OsCDPK13 accumulated in 2-week-old leaf sheath and callus, and became phosphorylated in response to cold and gibberellin (GA). OsCDPK13 gene expression and protein accumulation were up-regulated in response to GA₃ treatment, but suppressed in response to abscisic acid and brassinolide. Antisense OsCDPK13 transgenic rice lines were shorter than the vector control lines, and the expression of OsCDPK13 was lower in dwarf mutants of rice than in wild type. Furthermore, OsCDPK13 gene expression and protein accumulation were enhanced in response to cold, but suppressed under salt and drought stresses. Sense OsCDPK13 transgenic rice lines had higher recovery rates after cold stress than vector control rice. The expression of OsCDPK13 was stronger in cold-tolerant rice varieties than in cold-sensitive ones. The results suggest that OsCDPK13 might be an important signaling component in the response of rice to GA and cold stress.     

Proteomic analysis of rice leaf sheath during drought stress

Drought is one of the most severe limitations on the productivity of rainfed lowland and upland rice. To investigate the initial response of rice to drought stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to drought conditions from 2 to 6 days, and proteins were extracted from leaf sheaths, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. After drought stress for 2 to 6 days, 10 proteins increased in abundance and the level of 2 proteins decreased. The functional categories of these proteins were identified as defense, energy, metabolism, cell structure, and signal transduction. In addition to drought stress, accumulations of protein were analyzed under several different stress conditions. The levels of an actin depolymerizing factor, a light harvesting complex chain II, a superoxidase dismutase and a salt-induced protein were changed by drought and osmotic stresses, but not cold or salt stresses, or abscisic acid treatment. The effect of drought stress on protein in the leaf sheaths of drought-tolerant rice cultivar was also analyzed. The light harvesting complex chain II and the actin depolymerizing factor were present at high levels in a drought-tolerant rice cultivar before stress application. With drought stress, actin depolymerizing factor was expressed in leaf blades, leaf sheaths, and roots. These results suggest that actin depolymerizing factor is one of the target proteins induced by drought stress.     

Leaf contents differ depending on the position in a rice leaf sheath during sink-source transition.

Carbohydrate contents varied with position in a leaf sheath, and differed between the flag leaf sheath and the second leaf sheath below the flag leaf (-2 leaf sheath) in rice (Oryza sativa L.). In the -2 leaf sheath before heading, light microscopy revealed differences in the distribution of starch granules depending on position. Leaf sheaths were divided into several parts, and the contents of carbohydrates (starch, sucrose, and hexoses) were measured in each part. Before heading, the content of accumulated starch increased linearly from the top to the bottom in -2 leaf sheaths (r2=0.99, P<0.001), as did the contents of accumulated sucrose and hexoses in flag leaf sheaths (r2=0.94, P<0.01). In flag leaf sheaths, the relative content of sucrose synthase (SuS), which plays a central role in the degradation of sucrose into hexoses, increased from the top to the bottom, consistent with hexose contents. After heading, the accumulated carbohydrates were dramatically decreased. In -2 leaf sheaths, the activity of alpha-amylase (EC 3.2.1.1), the rate-limiting step in starch degradation, was consistent with the degree of starch degradation, but in flag leaf sheaths with little starch before heading. These results show that carbohydrate contents differ, depending on the position in a leaf sheath. In addition, there were big differences in leaf contents between flag leaf sheaths and -2 leaf sheaths.     

Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice

The mechanism of gibberellin (GA)-induced leaf sheath growth was examined using a dwarf mutant of rice (Oryza sativa L. cv. Tan-ginbozu) treated in advance with an inhibitor of GA biosynthesis. Gibberellic acid (GA₃) enhanced the growth of the second leaf sheath, but auxins did not. Measurement of the mitotic index and cell size revealed that cell elongation rather than cell division is promoted by GA₃. Gibberellic acid increased the extensibility of cell walls in the elongation zone of the leaf sheath. It also increased the total amount of osmotic solutes including sugars in the leaf sheath, but did not increase the osmotic concentration of the cell sap, due to an accompanying increase in cell volume by water absorption. In the later stage of GA₃-induced growth, starch granules completely disappeared from leaf sheath cells, whereas dense granules remained in control plants. These findings indicate that GA enhances cell elongation by increasing wall extensibility, osmotic concentration being kept unchanged by starch degradation. Received: 28 August 1997 / Accepted: 16 October 1997     

A proteomic approach to analyze salt-responsive proteins in rice leaf sheath

To examine the response of rice to salt stress, changes in protein expression were analyzed using a proteomic approach. To investigate dose- and time-dependent responses, rice seedlings were exposed to 50, 100 and 150 mM NaCl for 6 to 48 h. Proteins were extracted from leaf sheath and separated by two-dimensional polyacrylamide gel electrophoresis. Eight proteins showed 1- to 3-fold up-regulation in leaf sheath, in response to 50 mM NaCl for 24 h. Among these, three proteins were unidentified (LSY081, LSY262 and LSY363) while five proteins were identified as fructose bisphosphate aldolases, photosystem II (PSII) oxygen evolving complex protein, oxygen evolving enhancer protein 2 (OEE2) and superoxide dismutase (SOD). The maximum expression levels of seven proteins were at 24 h. Their expression declined after 48 h of 50 mM NaCl treatment. In contrast, SOD maintained its elevated expression throughout these conditions. The increased expression of proteins seen in the 50 mM NaCl treatment group was less pronounced in the groups receiving 100 or 150 mM NaCl for 24 h. The expression of SOD was a common response to cold, drought, salt and abscisic acid (ABA) stresses while the expression of LSY081, LSY363 and OEE2 was enhanced by salt and ABA stresses. LSY262 was expressed in leaf sheath and root, while fructose bisphosphate aldolases, PSII oxygen evolving complex protein and OEE2 were expressed in leaf sheath and leaf blade. LSY363 was expressed in leaf sheath but was below the level of detection in leaf blade and root. These results indicate that specific proteins expressed in specific regions of rice show a coordinated response to salt stress.     

Microtubule reorientation in shoots precedes bending during the gravitropic response of cut snapdragon spikes

The microtubule reorientation during the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes was investigated. Using indirect immunofluorescence methods, we examined changes in microtubule orientation in the cortex, endodermis and pith tissues of the shoot bending zone, in response to gravistimulation. Our results show that dense microtubule arrays were visible throughout the cortical, endodermal and pith shoot tissues, and that the transverse orientation of the microtubules (perpendicular to the growth axis) was specifically associated with the shoot growing bending zone. Microtubules showed gravity-induced kinetics of changes in their orientation, which occurred only in the upper stem flank and preceded shoot bending. While this observation, that the gravity-induced microtubule orientation precedes bending, was previously reported only in special above-ground organs such as coleoptiles and hypocotyls, our present study is the first to show that such patterns of change occur in mature flowering shoots. These changes were exhibited first in the upper flank of the cortex and then in the upper flank of the endodermis. No changes in microtubule orientation were observed in the cortex or endodermis tissues of the lower flanks or in the pith, suggesting that these tissues continue to grow during shoot gravistimulation. Our results imply that microtubules may be involved in growth cessation of the upper shoot flank occurring during the gravitropic bending of snapdragon cut spikes.     

Ntann12 annexin expression is induced by auxin in tobacco roots

Ntann12, encoding a polypeptide homologous to annexins, was found previously to be induced upon infection of tobacco with the bacterium Rhodococcus fascians. In this study, Ntann12 is shown to bind negatively charged phospholipids in a Ca(2+)-dependent manner. In plants growing in light conditions, Ntann12 is principally expressed in roots and the corresponding protein was mainly immunolocalized in the nucleus. Ntann12 expression was inhibited following plant transfer to darkness and in plants lacking the aerial part. However, an auxin (indole-3-acetic acid) treatment restored the expression of Ntann12 in the root system in dark conditions. Conversely, polar auxin transport inhibitors such as 1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid (TIBA) inhibited Ntann12 expression in light condition. These results indicate that the expression of Ntann12 in the root is linked to the perception of a signal in the aerial part of the plant that is transmitted to the root via polar auxin transport.     

Induction of systemic resistance in rice by leaf extracts of Datura metel against sheath blight disease

The leaf extracts of Datura metel [both aqueous leaf extract (ALE) and ethanolic leaf extract (ELE)] were observed here to find if they can induce systemic resistance in the rice commonly found in Eastern India. The results showed that after the treatment, the enzyme activities of all the defence-related enzymes increased to a certain level even without pathogenic infection in comparison with non-treated seedlings and then, maintain at constant level throughout the study period. When treated seedlings were infected with Rhizoctonia solani, the enzyme activities were increased more than in uninfected seedlings. The elevated enzyme activities gave the indication of an induced systemic resistance in rice. The ELE of D. metel showed better induction effect than ALE.     

Photosynthetic capacity is differentially affected by reductions in sedoheptulose-1,7-bisphosphatase activity during leaf development in transgenic tobacco plants

The impact of reduced sedoheptulose-1,7-bisphosphatase (SBPase) activity on photosynthetic capacity and carbohydrate status was examined during leaf expansion and maturation in antisense transgenic tobacco (Nicotiana tabacum L. cv Samsun) plants. In wild-type plants, photosynthetic capacity was lowest in young expanding leaves and reached a maximum in the fully expanded, mature leaves. In contrast, the transgenic antisense SBPase plants had the highest photosynthetic rates in the young expanding leaves and lowest rates in the mature leaves. In the mature, fully expanded leaves of the transgenic plants photosynthetic capacity was closely correlated with the level of SBPase activity. However, in the youngest leaves of the SBPase antisense plants, photosynthetic rates were close to, or higher than, those observed in wild-type plants, despite having a lower SBPase activity than the equivalent wild-type leaves. Reductions in SBPase activity affected carbohydrate levels in both the mature and young developing leaves. The overall trend was for decreased SBPase activity to lead to reductions in carbohydrate levels, particularly in starch. However, these changes in carbohydrate content were also dependent on the developmental status of the leaf. For example, in young expanding leaves of plants with the smallest reductions in SBPase activity, the levels of starch were higher than in wild-type plants. These data suggest that the source status of the mature leaves is an important determinant of photosynthetic development.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

Light interacts with auxin during leaf elongation and leaf angle development in young corn seedlings

Modern corn ( Zea mays L.) varieties have been selected for their ability to maintain productivity in dense plantings. We have tested the possibility that the physiological consequence of the selection of the modern hybrid, 3394, for increased crop yield includes changes in responsiveness to auxin and light. Etiolated seedlings in the modern line are shorter than in an older hybrid, 307, since they produce shorter coleoptile, mesocotyl, and leaves (blade as well as sheath). Etiolated 3394 seedlings, as well as isolated mesocotyl and sheath segments, were less responsive to auxin and an inhibitor of polar auxin transport, N-1-naphthylphthalamic acid (NPA). Reduced response of 3394 to auxin was associated with less reduction of elongation growth by light (white, red, far-red, blue) than in 307, whereas the activity of polar auxin transport (PAT) and its reduction by red or far-red light was similar in both genotypes. NPA reduced PAT in etiolated 3394 seedlings much less than in 307. A characteristic feature of 3394 plants is more erect leaves. In both hybrids, light (white, red, blue) increases leaf declination from the vertical, whereas NPA reduces leaf declination in 307, but not in 3394. Our results support findings that auxin and PAT are involved in elongation growth of corn seedlings, and we show that light interacts with auxin or PAT in regulation of leaf declination. We hypothesize that, relative to 307, more erect leaves in the modern hybrid may be primarily a consequence of a reduced amount of auxin receptor(s) and reduced responsiveness to light in etiolated 3394 plants. The more erect leaves in 3394 may contribute to the tolerance of the modern corn hybrid to dense planting.     

Leaf respiration is differentially affected by leaf vs. stand-level night-time warming

Plant respiration is an important physiological process in the global carbon cycle serving as a major carbon flux from the biosphere to the atmosphere. Respiration is sensitive to temperature providing a link between environmental variability, climate change and the global carbon cycle. We measured leaf respiration in Populus deltoides after manipulating the air temperature surrounding part of a single leaf, and compared this to the temperature response of the same leaves after manipulating the temperature of the stand. The short‐term temperature response of respiration (Q₁₀– change in the respiration rate with a 10 °C increase in leaf temperature) was 1.7 when the leaf temperature was manipulated, but 2.1 when the stand‐level temperature was changed. As a result, total night‐time carbon release during the five‐day experiment was 21% lower when using the Q₁₀ estimates from the tradition leaf manipulation compared to the stand‐level manipulation. We conclude that the temperature response of leaf respiration is related to whole plant carbon and energy demands, and that appropriate experimental procedures are required in examining respiratory CO₂ release under variable temperature conditions.     

Nicotine synthesis in Nicotiana tabacum L. induced by mechanical wounding is regulated by auxin

The effects of different kinds of mechanical wounding on nicotine production in tobacco plants were compared, with sand or hydroponics culture under controlled conditions. Both removal of the shoot apex and damage of the youngest unfolded leaves nos 1 and 2 by a comb-like brusher with 720 punctures caused an increase in nicotine concentration in whole plants at day 3, and reached its highest level at day 6. The nicotine concentration induced by excision of the shoot apex was much higher than that induced by leaf wounding. Both treatments also caused an increase in jasmonic acid (JA) concentration within 90 min in the shoot, followed by an increase in the roots (210 min), in which the JA concentration induced by leaf wounding was significantly higher than that induced by excision of the shoot apex. The increase in nicotine concentration occurred throughout the whole plant, especially in the shoot, while the increase in JA concentration in the shoot was restricted to the damaged tissues, and was not observed in the adjacent tissues. Removal of the lateral buds that emerged after excision of the shoot apex caused a further increase in nicotine concentrations in the plant tissues. Removal of mature leaves, however, did not cause any changes in nicotine concentration in the plant, even though the degree of wounding in this case was comparable with that occurring with apex removal. The results suggest that the nicotine production in tobacco plants was not correlated with the degree of wounding (cut-surface or punctures), but was highly dependent on the removal of apical meristems and hence on the major sources of auxin in the plant. Furthermore, immediate application of 1-naphthylacetic acid (NAA) on the cut surface after removing the shoot apex completely inhibited the increase both in nicotine in whole plants and in JA in the damaged stem segment and roots. Application of an auxin transport inhibitor around the stem directly under the shoot apex of intact plants also caused an increase in nicotine concentration in the whole plant. The results strongly suggest that auxin serves as a negative signal to regulate nicotine synthesis in roots of tobacco plants.     

Photosynthesis of the flag leaf blade and its sheath in high-yielding hybrid rice 'Liangyoupeijiu'.

Using high-yielding hybrid rice 'Liangyoupeijiu' (LYP9) and hybrid rice 'Shanyou 63' (SY63) as the experimental materials and using (14)C radio-autography, the photosynthetic capacities and distribution of photosynthates in flag leaf blades and sheaths of LYP9 were studied. The results showed that net photosynthetic rates (Pn) of the flag leaf blades and sheaths of LYP9 were much higher than those of SY63; the light transmissivity rates (LT) measured at the medium height of the flag leaf sheaths and the penultimate leaf sheaths were also significantly higher than those of SY63. The incipient activities, total activities and activation percentages of Rubisco in the flag leaf blade and sheath of LYP9 were all higher than those of SY63. The photosynthate transport rate in the sheaths of LYP9, and the quantity of photosynthate transported to the spikes and transformed to economic yield of LYP9 were all higher than those of SY63. The photosynthates produced by the sheaths were mainly transported to spike to make a certain contribution (about 15%) to yield.     

Actin expression is induced and three isoforms are differentially expressed during germination in Zea mays

Previous analysis of actin in a dicotyledonous plant, Phaseolus vulgaris (or common bean), showed very low actin levels in cotyledons but they were concentrated in the embryo axis. Upon imbibition, actin expression increased 5-fold and a maximum of four actin isoforms were observed, two of them transient and two major ones were steadily expressed. In this work, analysis of the actin expression in a monocotyledonous plant, Zea mays (or maize), and over a longer period of germination/growth, showed that striking similarities exist. Actin is present in all the seed components, but it is mainly concentrated in the embryo axis. The expression of maize actin was induced during post-imbibition at both the protein and mRNA levels. Sharp increases in actin appeared from 24-48 h and again from 72-96 h. A more modest and steady actin mRNA increase in expression was observed; however, it did not appear as dramatic as in the case of common bean due to the presence of readily detectable amounts of message in the dry maize seed. The isoform distribution in the dry seed showed a pattern of at least three isovariants of pIs approximately 5.0, 5.1, and 5.2, which were differentially expressed at the various post-imbibition times analysed. Two of the actin isoforms at 48 h post-imbibition cross-reacted with a phosphotyrosine-specific antibody and they are the product of three expressed genes as shown by in vitro translation assays. These data indicate that maize actin protein and mRNA expression is induced upon the trigger of germination, and the isoform expression kinetics and patterns resemble those from bean, suggesting that, in both species, actin expression at these early germination/growth stages is a highly regulated event.     

CsPLDalpha1 and CsPLDgamma1 are differentially induced during leaf and fruit abscission and diurnally regulated in Citrus sinensis

Understanding leaf and fruit abscission is essential in order to develop strategies for controlling the process in fruit crops. Mechanisms involved in signalling leaf and fruit abscission upon induction by abscission agents were investigated in Citrus sinensis cv. 'Valencia'. Previous studies have suggested a role for phospholipid signalling; hence, two phospholipase D cDNA sequences, CsPLDalpha1 and CsPLDgamma1, were isolated and their role was examined. CsPLDalpha1 expression was reduced in leaves but unaltered in fruit peel tissue treated with an ethylene-releasing compound (ethephon), or a fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP). By contrast, CsPLDgamma1 expression was up-regulated within 6 h (leaves) and 24 h (fruit peel) after treatment with ethephon or CMNP, respectively. CsPLDalpha1 expression was diurnally regulated in leaf blade but not fruit peel. CsPLDgamma1 exhibited strong diurnal oscillation in expression in leaves and fruit peel with peak expression around midday. While diurnal fluctuation in CsPLDalpha1 expression appeared to be light-entrained in leaves, CsPLDgamma1 expression was regulated by light and the circadian clock. The diurnal expression of both genes was modulated by ethylene-signalling. The ethephon-induced leaf abscission and the ethephon- and CMNP-induced decrease in fruit detachment force were enhanced by application during rising diurnal expression of CsPLDgamma1. The results indicate differential regulation of CsPLDalpha1 and CsPLDgamma1 in leaves and fruit, and suggest possible roles for PLD-dependent signalling in regulating abscission responses in citrus.     

Peroxidase genes differentially respond to auxin during the formation of adventitious roots in soybean hypocotyl

In our investigation, auxins (IAA, IBA and NAA) effectively promoted rooting in soybean hypocotyls. The activity of anionic peroxidase (POX) (pI 3.7) and cationic POX (pI 8.5) was significantly suppressed by exogenous auxins on day 2 (the inductive phase). Some particular anionic POXs (pI 4.0 and pI 5.3) significantly increased in IBA-treated tissues as compared with the control when the incubation time was prolonged to day 3 and day 4 (the initiation phase). We sequenced 5′-flanking region of pI 8.5 and pI 5.3 POX genes using the PLACE and PlantCARE databases to identify several potential cis-regulatory elements. The pI 8.5 POX gene promoter contained two sites that were homologous to sequences commonly found in auxin response elements; motifs ARF/AuxRE and CATATGGMSAUR. During the inductive phase, the activity of pI 8.5 POX was significantly suppressed by the exogenously applied auxins. The pI 8.5 POX gene promoter contained both ARF/AuxRE and CATATGGMSAUR motifs that responded to auxins earlier than the pI 5.3 POX gene. Hence, the pI 8.5 POX gene might belong to primary auxin response genes. The pI 5.3 POX gene, which responded to auxins a day or two later, contained only ARF/AuxRE motif. Moreover, unlike pI 8.5 and pI 3.7 POXs that were suppressed by auxins, the pI 5.3 POX was induced or enhanced by the applied auxins, especially IBA. The pI 5.3 POX might generate H₂O₂ which caused the auxin-induced growth at the initiation phase during the formation of adventitious root in soybean hypocotyls.