Expression of the Arabidopsis Mg-chelatase H subunit alleviates iron deficiency-induced stress in transgenic rice.

The most common symptom of iron (Fe) deficiency in plants is leaf chlorosis caused by impairment of chlorophyll biosynthesis. Magnesium (Mg)-chelatase H subunit (CHLH) is a key component in both chlorophyll biosynthesis and plastid signaling, but its role in Fe deficiency is poorly understood. Heterologous expression of the Arabidopsis thaliana Mg-chelatase H subunit gene (AtCHLH) increased Mg-chelatase activity by up to 6-fold and abundance of its product, Mg-protoporphyrin IX (Mg-Proto IX), by 60-75% in transgenic rice (Oryza sativa) seedlings compared to wild-type (WT) controls. Noticeably, the transgenic seedlings showed alleviation of Fe deficiency symptoms, as evidenced by their less pronounced leaf chlorosis and lower declines in shoot growth, chlorophyll contents, and photosynthetic efficiency, as indicated by F v/F m and electron transport rate, compared to those in WT seedlings under Fe deficiency. Porphyrin metabolism was differentially regulated by Fe deficiency between WT and transgenic seedlings, particularly with a higher level of Mg-Proto IX in transgenic lines, showing that overexpression of AtCHLH reprograms porphyrin metabolism in transgenic rice. Leaves of Fe-deficient transgenic seedlings exhibited greater upregulation of deoxymugineic acid biosynthesis-related genes (i.e., NAS, NAS2, and NAAT1), YSL2 transporter gene, and Fe-related transcription factor genes IRO2 and IDEF2 than those of WT, which may also partly contribute to alleviating Fe deficiency. Although AtCHLH was postulated to act as a receptor for abscisic acid (ABA), exogenous ABA did not alter the phenotypes of Fe-deficient WT or transgenic seedlings. Our study demonstrates that modulation of porphyrin biosynthesis through expression of AtCHLH in transgenic rice alleviates Fe deficiency-induced stress, suggesting a possible role for CHLH in Fe deficiency responses.

Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis.

Senescence is controlled by time-evolving networks that describe the temporal transition of interactions among senescence regulators. Here, we present time-evolving networks for NAM/ATAF/CUC (NAC) transcription factors in Arabidopsis during leaf aging. The most evident characteristic of these time-dependent networks was a shift from positive to negative regulation among NACs at a presenescent stage. ANAC017, ANAC082, and ANAC090, referred to as a "NAC troika," govern the positive-to-negative regulatory shift. Knockout of the NAC troika accelerated senescence and the induction of other NACs, whereas overexpression of the NAC troika had the opposite effects. Transcriptome and molecular analyses revealed shared suppression of senescence-promoting processes by the NAC troika, including salicylic acid (SA) and reactive oxygen species (ROS) responses, but with predominant regulation of SA and ROS responses by ANAC090 and ANAC017, respectively. Our time-evolving networks provide a unique regulatory module of presenescent repressors that direct the timely induction of senescence-promoting processes at the presenescent stage of leaf aging.

Differential Antioxidant Responses and Perturbed Porphyrin Biosynthesis after Exposure to Oxyfluorfen and Methyl Viologen in Oryza sativa.

We compared antioxidant responses and regulation of porphyrin metabolism in rice plants treated with oxyfluorfen (OF) or methyl viologen (MV). Plants treated with MV exhibited not only greater increases in conductivity and malondialdehyde but also a greater decline in Fv/Fm, compared to plants treated with OF. MV-treated plants had greater increases in activities of superoxide dismutase (SOD) and catalase (CAT) as well as transcript levels of SODA and CATA than OF-treated plants after 28 h of the treatments, whereas increases in ascorbate peroxidase (APX) activity and transcript levels of APXA and APXB were greater in OF-treated plants. Both OF- and MV-treated plants resulted in not only down-regulation of most genes involved in porphyrin biosynthesis but also disappearance of Mg-porphyrins during the late stage of photooxidative stress. By contrast, up-regulation of heme oxygenase 2 (HO2) is possibly part of an efficient antioxidant response to compensate photooxidative damage in both treatments. Our data show that down-regulated biosynthesis and degradation dynamics of porphyrin intermediates have important roles in photoprotection of plants from perturbed porphyrin biosynthesis and photosynthetic electron transport. This study suggests that porphyrin scavenging as well as strong antioxidative activities are required for mitigating reactive oxygen species (ROS) production under photooxidative stress caused by OF and MV.

Increased expression of Fe-chelatase leads to increased metabolic flux into heme and confers protection against photodynamically induced oxidative stress.

Fe-chelatase (FeCh, EC 4.99.1.1) inserts Fe(2+) into protoporphyrin IX (Proto IX) to form heme, which influences the flux through the tetrapyrrole biosynthetic pathway as well as fundamental cellular processes. In transgenic rice (Oryza sativa), the ectopic expression of Bradyrhizobium japonicum FeCh protein in cytosol results in a substantial increase of FeCh activity compared to wild-type (WT) rice and an increasing level of heme. Interestingly, the transgenic rice plants showed resistance to oxidative stress caused not only by the peroxidizing herbicide acifluorfen (AF) as indicated by a reduced formation of leaf necrosis, a lower conductivity, lower malondialdehyde and H2O2 contents as well as sustained Fv/Fm compared to WT plants, but also by norflurazon, paraquat, salt, and polyethylene glycol. Moreover, the transgenic plants responded to AF treatment with markedly increasing FeCh activity. The accompanying increases in heme content and heme oxygenase activity demonstrate that increased heme metabolism attenuates effects of oxidative stress caused by accumulating porphyrins. These findings suggest that increases in heme levels and porphyrin scavenging capacity support a detoxification mechanism serving against porphyrin-induced oxidative stress. This study also implicates heme as possibly being a positive signal in plant stress responses.

Porphyrin biosynthesis control under water stress: sustained porphyrin status correlates with drought tolerance in transgenic rice.

A controlled flow of porphyrin metabolites is critical for organisms, but little is known about the control of porphyrin biosynthesis under environmental stress. We monitored transgenic rice (Oryza sativa) plants expressing Myxococcus xanthus protoporphyrinogen oxidase (PPO) for their response to drought stress. Transgenic plants showed significantly improved drought tolerance, as indicated by a higher shoot water potential, less oxidative damage, and a more favorable redox balance compared with wild-type plants. Both transgenic and wild-type plants responded to the onset of drought stress, even prior to changes in shoot water potential and oxidative metabolism, by drastically scavenging porphyrin intermediates in leaves, which was crucial for alleviating reactive oxygen species-induced stress. Protoporphyrin IX, protochlorophyllide, magnesium-protoporphyrin IX, and its methyl ester were absent or hardly detected with the intensification of water stress (-3.1 MPa) in the wild type, whereas transgenic plants retained these intermediates to some extent. Additionally, the expression and activity of most enzymes involved in porphyrin biosynthesis, particularly in the chlorophyll branch, were primarily down-regulated under dehydrating conditions, with stronger repression in the wild type than in transgenic plants. There was up-regulation of Glutamate 1-Semialdehyde Aminotransferase, PPO1, and Fe Chelatase2 transcripts in drought-stressed transgenic plants, enabling the transgenic plants to make larger pools of 5-aminolevulinic acid and protoporphyrin IX available for subsequent steps in the heme branch. Overexpression of PPO ultimately protected the transgenic plants from drought-induced cytotoxicity, demonstrating clearly that manipulation of porphyrin biosynthesis can produce drought-tolerant plants. Our results support a possible role for tetrapyrroles in signaling their metabolic state and in plant protection under drought stress conditions.