Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae

Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map‐based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST–OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower‐related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild‐type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blast‐induced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus.     

The jasmonate-free rice mutant hebiba is affected in the response of phyA'/phyA" pools and protochlorophyllide biosynthesis to far-red light.

Phytochrome (phy) A in its two native isoforms (phyA' and phyA") and the active (Pchlide(655)) and inactive (Pchlide(633)) protochlorophyllides were investigated by low-temperature fluorescence spectroscopy in the tips of rice (Oryza sativa L. Japonica cv Nihonmasari) coleoptiles from wild type (WT) and the jasmonate-deficient mutant hebiba. The seedlings were either grown in the dark or under pulsed (FRp) or continuous (FRc) far-red light (lambda(a) >/= 720 nm) of equal fluences. In the dark, the mutant had a long mesocotyl and a short coleoptile, whereas the situation was reversed under FR: short mesocotyl and long coleoptile, suggesting that the effect is mediated by phyA. Under these conditions the WT displayed a short coleoptile and emergence of the first leaf. In the dark, the spectroscopic and photochemical properties of phyA, its content and the proportion of its two pools, phyA' and phyA", were virtually identical between WT and hebiba. However, the total content of protochlorophyllides was higher in the mutant. Upon illumination with FRc, [phyA] declined in the WT and the ratio between phyA' and phyA" shifted towards phyA". In hebiba, the light-induced decline of [phyA] was less pronounced and the ratio between phyA' and phyA" did not shift. Moreover, in the WT, FRp stimulated the biosynthesis of Pchlide(655), whereas FRc was inhibiting. In contrast, in the mutant, both FRp and FRc stimulated the synthesis of Pchlide(655). This means that FRc caused the opposite effect in hebiba. This difference correlates with a slower photodestruction of primarily the light-labile phyA' pool in hebiba.     

The roles of phytochromes in elongation and gravitropism of roots

Gravitropic orientation and the elongation of etiolated hypocotyls are both regulated by red light through the phytochrome family of photoreceptors. The importance of phytochromes A and B (phyA and phyB) in these red light responses has been established through studies using phy mutants. To identify the roles that phytochromes play in gravitropism and elongation of roots, we studied the effects of red light on root elongation and then compared the gravitropic curvature from roots of phytochrome mutants of Arabidopsis (phyA, phyB, phyD and phyAB) with wild type. We found that red light inhibits root elongation approximately 35% in etiolated seedlings and that this response is controlled by phytochromes. Roots from dark- and light-grown double mutants (phyAB) and light-grown phyB seedlings have reduced elongation rates compared with wild type. In addition, roots from these seedlings (dark/light-grown phyAB and light-grown phyB) have reduced rates of gravitropic curvature compared with wild type. These results demonstrate roles for phytochromes in regulating both the elongation and gravitropic curvature of roots.     

Interaction between two auxin-resistant mutants and their effects on lateral root formation in rice (Oryza sativa L.)

Since root elongation is very sensitive to auxin, screening for reduced inhibition in root elongation has been an important method for the detection of auxin-resistant mutants. Two recessive auxin-resistant lines of rice (Oryza sativa L. ssp. indica cv. IR8), arm1 and arm2, have been isolated by screening for resistance to 2,4-dichlorophenoxyacetic acid (2,4-D). arm1 displays a variety of morphological defects including reduced lateral root formation, increased seminal root elongation, reduced root diameter, and impaired xylem development in roots, while the arm2 phenotype is almost similar to wild-type IR8 except for a slightly reduced lateral root formation, impaired xylem development in roots and an enhanced plant height. Although the growth of arm2 roots exhibited a resistance to 2,4-D, it was sensitive to 1-naphthaleneacetic acid (NAA) as the wild type. At the same time, the arm2 roots showed a reduced [14C]2,4-D uptake while uptake of [3H]NAA was normal, suggesting that the resistance to 2,4-D of arm2 roots is due to a defect in 2,4-D uptake. To investigate the possible interaction between arm1 and arm2 genes, a double mutant has been constructed. The roots of arm1 arm2 double mutant were more resistant to 2,4-D and formed fewer lateral roots than those of either single mutant, suggesting that the two genes show synergistic effects with respect to both auxin response and lateral root formation. By contrast, all these mutants displayed the normal gravitropic response in roots, as did the wild-type plants. Taken together, Arm1 and Arm2 genes seem to function in different processes in the auxin-response pathways leading to lateral root formation.     

The growth of tomato (Lycopersicon esculentum Mill.) hypocotyls in the light and in darkness differentially involves auxin.

Light and auxin antagonistically regulate hypocotyl elongation. We have investigated the physiological interactions of light and auxin in the control of tomato (Lycopersicon esculentum Mill.) hypocotyl elongation by studying the auxin-insensitive mutant diageotropica (dgt). The length of the hypocotyls of the dgt mutant is significantly reduced when compared to the wild type line Ailsa Craig (AC) in the dark and under red light, but not under the other light conditions tested, indicating that auxin sensitivity is involved in the elongation of hypocotyls only in these conditions. Similarly, the auxin transport inhibitor naphthylphthalamic [correction of naphtylphtalamic] acid (NPA) differentially affects elongation of dark- or light-grown hypocotyls of the MoneyMaker (MM) tomato wild type. Using different photomorphogenic mutants, we demonstrate that at least phytochrome A, phytochrome B1 and, to a much lesser extent [correction of extend], cryptochrome 1, are necessary for a switch from an auxin transport-dependent elongation of hypocotyls in the dark to an auxin transport-independent elongation in the light. Interestingly, the dgt mutant and NPA-treated seedlings exhibit a looped phenotype only under red light, indicating that the negative gravitropism of hypocotyls also differentially involves auxin in the various light conditions.     

Inhibition of arabidopsis hypocotyl elongation by jasmonates is enhanced under red light in phytochrome B dependent manner

Jasmonates are phytohormones derived from oxygenated fatty acids that regulate a broad range of plant defense and developmental processes. In Arabidopsis, hypocotyl elongation under various light conditions was suppressed by exogenously supplied methyl jasmonate (MeJA). Moreover, this suppression by MeJA was particularly effective under red light condition. Mutant analyses suggested that SCF^COI1-mediated proteolysis was involved in this function. However, MeJA action still remained in the coi1 mutant, and (+)-7-iso-JA-L-Ile, a well-known active form of jasmonate, had a weaker effect than MeJA under the red light condition, suggesting that unknown signaling pathway are present in MeJA-mediated inhibition of hypocotyl elongation. EMS mutant screening identified two MeJA-insensitive hypocotyl elongation mutants, jasmonate resistance long hypocotyl 1 (jal1) and jal36, which had mutations in the phytochrome B (PHYB) gene. These analyses suggested that inhibition of hypocotyl elongation by jasmonates is enhanced under red light in phyB dependent manner.     

Isolation and characterization of rice phytochrome A mutants

To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception--far-red light-dependent high irradiance response and very low fluence response--and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings.     

GER1, a GDSL motif-encoding gene from rice is a novel early light- and jasmonate-induced gene

The reaction of the rice mutant HEBIBA differs from that of wild-type rice in that the mutant responds inversely to red light and is defective in the light-triggered biosynthesis of jasmonic acid (JA). Using the wild type and the HEBIBA mutant of rice in a differential display screen, we attempted to identify genes that act in or near the convergence point of light and JA signalling. We isolated specifically regulated DNA fragments from approximately 10 000 displayed bands, and identified a new early light- and JA-induced gene. This gene encodes an enzyme containing a GDSL motif, showing 38 % identity at the amino acid level to lipase Arab-1 in Arabidopsis thaliana. The GDSL CONTAINING ENZYME RICE 1 gene (GER1) is rapidly induced by both red (R) and far-red (FR) light and by JA. The results are discussed with respect to a possible role for GER1 as a negative regulator of coleoptile elongation in the context of recent findings on the impact of JA on light signalling.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

A Temporarily Red Light-Insensitive Mutant of Tomato Lacks a Light-Stable,B-Like Phytochrome

We have selected four recessive mutants in tomato (Lycopersicon esculentum Mill.) that, under continuous red light (R), have long hypocotyls and small cotyledons compared to wild type (WT), a phenotype typical of phytochrome B (phyB) mutants of other species. These mutants, which are allelic, are only insensitive to R during the first 2 days upon transition from darkness to R, and therefore we propose the gene symbol tri (temporarily red light insensitive). White light-grown mutant plants have a more elongated growth habit than that of the WT. An immunochemically and spectrophotometrically detectable phyB-like polypeptide detectable in the WT is absent or below detection limits in the tri1 mutant. In contrast to the absence of an elongation growth response to far-red light (FR) given at the end of the daily photoperiod (EODFR) in all phyB-deficient mutants so far characterized, the tri1 mutant responds to EODFR treatment. The tri1 mutant also shows a strong response to supplementary daytime far-red light. We propose that the phyB-like phytochrome deficient in the tri mutants plays a major role during de-etiolation and that other light-stable phytochromes can regulate the EODFR and shade-avoidance responses in tomato.     

Light-stimulated cotyledon expansion in the blu3 and hy4 mutants of Arabidopsis thaliana.

Cotyledon expansion in response to blue light was compared for wild-type Arabidopsis thaliana (L.) Heynh. and the mutants blu3 and hy4, which show reduced inhibition of hypocotyl growth in blue light. White, blue, and red light stimulated cotyledon expansion in both intact and excised cotyledons of wild-type seedlings (ecotypes No-0, WS, Co-0, La-er). Cotyledons on intact blu3 and hy4 seedlings did not grow as well as those on the wild type in response to blue light, but pretreatment of blu3 seedlings with low fluence rates of red light increased their responsiveness to blue light. Excision of cotyledons alleviated the mutant phenotype so that both mutant and wild-type cotyledons grew equally well in blue light. The loss of the mutant cotyledon phenotype upon excision indicates that the blu3 and hy4 lesions affect cotyledon expansion indirectly via a whole-plant response to light. Furthermore, the ability of excised, mutant cotyledons to grow normally in blue light shows that this growth response to blue light is mediated by a photosystem other than the ones impaired by the blu3 and hy4 lesions.     

Arabidopsis Mutants Lacking Blue Light-Dependent Inhibition of Hypocotyl Elongation

We have isolated a new class of photomorphogenic mutants in Arabidopsis. Hypocotyl elongation is not inhibited in the mutant seedlings by continuous blue light but is inhibited by far red light, indicating that these mutations are phenotypically different from the previously isolated long hypocotyl (hy) mutants. Complementation analysis indicated that recessive nuclear mutations at three genetic loci, designated blu1, blu2, and blu3, can result in the blu mutant phenotype and that these mutants are genetically distinct from other long hypocotyl mutants. The BLU genes appear to be important only during seedling development because the blu mutations have little effect on mature plants, whereas hypocotyl elongation and cotyledon expansion are altered in seedlings. The genetic separation of the blue and far red sensitivities of light-induced hypocotyl inhibition in the blu and hy mutants demonstrates that two photosensory systems function in this response.     

Photophysiology and phytochrome content of long-hypocotyl mutant and wild-type cucumber seedlings

Photomorphogenetic responses have been studied in a cucumber (Cucumis sativus L.) mutant (lh), which has long hypocotyls in white light (WL). While etiolated seedlings of this mutant have a similar phytochrome content and control of hypocotyl elongation as wild type, deetiolation is retarded and WL-grown seedlings show reduced phytochrome control. Spectrophotometric measurements exhibit that WL-grown tissues of the lh mutant (flower petals and Norflurazon-bleached leaves) contain 35 to 50% of the phytochrome level in the wild type. We propose that this is a consequence of a lack of light-stable phytochrome, in agreement with our hypothesis proposed on the basis of physiological experiments. The lh mutant lacks an end-of-day far-red light response of hypocotyl elongation. This enables the end-of-day far-red light response, clearly shown by the wild type, to be ascribed to the phytochrome, deficient in the lh mutant. Growth experiments in continuous blue light (BL) and continuous BL + red light (RL) show that when RL is added to BL, hypocotyl growth remains inhibited in the wild type, whereas the lh mutant exhibits significant growth promotion compared to BL alone. It is proposed that the hypocotyls fail to grow long in low fluence rate BL because photosynthesis is insufficient to sustain growth.     

Light-induced stomatal movement of selected Arabidopsis thaliana mutants

Various Arabidopsis thaliana mutants with defects in phytohormone signal transduction or the reception of light were analysed with regard to their stomatal response in a red, red/blue light irradiation programme. Stomatal response to light was detected with a customized gas exchange measurement device, optimized for the small model plant. Small transpiration-kinetic variations of the two wild-type lines Columbia (Col) and Landsberg erecta (Ler) were observed. A comparison of the mutant lines to the respective wild type revealed significant differences for the phytochrome A (phyA-103), the abscisic acid insensitive (aba3-2) and the auxin resistant (axr1-3) mutant. Furthermore, the zeaxanthin-less mutant line npq1-2 showed no alterations in stomatal response to light.     

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.     

New lv Mutants of Pea Are Deficient in Phytochrome B

The lv-1 mutant of pea (Pisum sativum L.) is deficient in responses regulated by phytochrome B (phyB) in other species but has normal levels of spectrally active phyB. We have characterized three further lv mutants (lv-2, lv-3, and lv-4), which are all elongated under red (R) and white light but are indistinguishable from wild type under far-red light. The phyB apoprotein present in the lv-1 mutant was undetectable in all three new lv mutants. The identification of allelic mutants with and without phyB apoprotein suggests that Lv may be a structural gene for a B-type phytochrome. Furthermore, it indicates that the lv-1 mutation results specifically in the loss of normal biological activity of this phytochrome. Red-light-pulse and fluence-rate-response experiments suggest that lv plants are deficient in the low-fluence response (LFR) but retain a normal very-low-fluence-rate-dependent response for leaflet expansion and inhibition of stem elongation. Comparison of lv alleles of differing severity indicates that the LFR for stem elongation can be mediated by a lower level of phyB than the LFR for leaflet expansion. The retention of a strong response to continuous low-fluence-rate R in all four lv mutants suggests that there may be an additional phytochrome controlling responses to R in pea. The kinetics of phytochrome destruction and reaccumulation in the lv mutant indicate that phyB may be involved in the light regulation of phyA levels.     

拟南芥PRRs基因在红光和蓝光信号转导中的作用初探

以拟南芥为材料,在红光和蓝光下对PRRs(pseudo-response regulators)突变体prr5p、rr7、prr9和toc1及其野生型的下胚轴表型进行比较观察,并采用实时定量PCR方法对突变体中光信号通路相关基因ZTL(zeitlupe)和CO(constans)的节律表达进行分析.结果表明:在红光下,prr5和toc1的下胚轴长度比野生型显著增长,在蓝光下,prr7p、rr9和toc1较野生型短,表明突变体降低了拟南芥对红光的敏感性,却增强了对蓝光的敏感性.红光和蓝光下,PRRs突变体中ZTL和CO的mRNA节律表达与野生型明显不同,其中红光下prr5和prr7、蓝光下prr5和toc1中的ZTLmRNA的表达显著下降且节律消失;红光下prr7和prr9以及蓝光下prr5突变体中的COmRNA表现基本无节律.因此推测,PRRs与ZTL的相互作用很可能在红光和蓝光信号转导途径中发挥作用,且PRRs基因极有可能参与了红光和蓝光对CO的调控.