Chloroplast-localized iron superoxide dismutases FSD2 and FSD3 are functionally distinct in Arabidopsis.

Superoxide dismutases (SODs) protect against reactive oxygen species (ROS) by detoxifying superoxide. Three types of SOD are present in plants: FeSOD, CuSOD, and MnSOD. The Arabidopsis thaliana genome contains three FeSOD genes, in which two (FSD2, and FSD3) are targeted to chloroplast thylakoids. Loss of FSD2 or FSD3 expression impairs growth and causes leaf bleaching. FSD2 and FSD3 form heterocomplexes present in chloroplast nucleoids, raising the question of whether FSD2 and FSD3 are functionally interchangeable. In this study, we examined how loss of FSD2 or FSD3 expression affects photosynthetic processes and whether overexpression of one compensates for loss of the other. Whereas loss of the cytosolic FSD1 had little effect, an fsd2 mutant exhibited increased superoxide production, reduced chlorophyll levels, lower PSII efficiency, a lower rate of CO2 assimilation, but elevated non-photochemical quenching (NPQ). In contrast, fsd3 mutants failed to survive beyond the seedling stage and overexpression of FSD2 could not rescue the seedlings. Overexpression of FSD3 in an fsd2 mutant, however, partially reversed the fsd2 mutant phenotype resulting in improved growth characteristics. Overexpression of FSD2 or FSD3, either individually or together, had little effect. These results indicate that, despite functioning as FeSODs, FSD2 and FSD3 are functionally distinct.

Plant growth and fertility requires functional interactions between specific PABP and eIF4G gene family members.

The initiation of protein synthesis requires the involvement of the eukaryotic translation initiation factor (eIF) 4G to promote assembly of the factors needed to recruit a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, those in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization. Species of the Brassicaceae and the Cleomaceae also express a divergent eIFiso4G isoform, referred to as eIFiso4G2, not found elsewhere in the plant kingdom. Despite their divergence, eIF4G and eIFiso4G interact with eIF4A, eIF4B, and eIF4E isoforms needed for binding an mRNA. eIF4G and eIFiso4G also interact with the poly(A)-binding protein (PABP) which promotes ribosome recruitment to an mRNA. Increasing the complexity of such an interaction, however, Arabidopsis also expresses three PABP isoforms (PAB2, PAB4, and PAB8) in vegetative and reproductive tissues. In this study, the functional interactions among the eIF4G and the widely-expressed PABP isoforms were examined. Loss of PAB2 or PAB8 in combination with loss of eIF4G or eIFiso4G had little to no effect on growth or fertility whereas pab2 pab8 eif4g or pab2 pab8 eifiso4g1/2 mutants exhibited smaller stature and reduced fertility. Although the pab4 eifiso4g1 mutant grows normally and is fertile, pab4 eif4g or pab4 eifiso4g2 mutants could not be isolated. Even pab4/PAB4 eif4g/eIF4G heterozygous plants exhibited growth defects and low fertility. Mutant co-inheritance analysis in reciprocal crosses with wild-type plants revealed that most ovaries and pollen from pab4/PAB4 eif4g/eIF4G plants were PAB4 eif4g. Similarly, co-inheritance studies with pab4/PAB4 eifiso4g2/eIFiso4G2 plants suggested most ovaries were PAB4 eifiso4g2. These results suggest that a functional interaction between PAB4 and eIF4G and between PAB4 and eIFiso4G2 is required for growth and normal fertility.

Class II members of the poly(A) binding protein family exhibit distinct functions during Arabidopsis growth and development.

The poly(A)-binding protein (PABP) binds to the poly(A) tail of eukaryotic cellular mRNAs and contributes to their stability and translational efficiency. In plants, PABP is expressed from an unusually large gene family grouped into 3 classes that expanded during the evolution of land plants. Subsequent to expansion of the family, members diverged in their primary sequence and in expression. Further expansion of the family and divergence of its members in the Brassicaceae demonstrate the continued dynamic evolution of PABP in plants. In this study, the function of the widely-expressed class II PABP family members was examined to determine how individual class II members contribute to plant growth and development. Of the 3 class II PABP members, PAB2 and PAB4 contribute most to vegetative growth and vegetative-to-floral transition whereas PAB2, and the recently-evolved third class II member, PAB8, contribute to inflorescence and silique growth. Interestingly, although class I and class III PABP members are expressed specifically in reproductive organs, class II PABP members are also necessary for fertility in that the combinatorial loss of PAB2 and either PAB4 or PAB8 expression resulted in reduced fertility. Although all 3 class II members are required for protein expression, PAB4 contributes most to the steady-state level of a reporter mRNA and to protein expression. These findings suggest that class II PABP members are partially overlapping in function but also involved in distinct aspects of plant growth and development.

Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis.

The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5'-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation.

Ethylene Regulates Energy-Dependent Non-Photochemical Quenching in Arabidopsis through Repression of the Xanthophyll Cycle.

Energy-dependent (qE) non-photochemical quenching (NPQ) thermally dissipates excess absorbed light energy as a protective mechanism to prevent the over reduction of photosystem II and the generation of reactive oxygen species (ROS). The xanthophyll cycle, induced when the level of absorbed light energy exceeds the capacity of photochemistry, contributes to qE. In this work, we show that ethylene regulates the xanthophyll cycle in Arabidopsis. Analysis of eto1-1, exhibiting increased ethylene production, and ctr1-3, exhibiting constitutive ethylene response, revealed defects in NPQ resulting from impaired de-epoxidation of violaxanthin by violaxanthin de-epoxidase (VDE) encoded by NPQ1. Elevated ethylene signaling reduced the level of active VDE through decreased NPQ1 promoter activity and impaired VDE activation resulting from a lower transthylakoid membrane pH gradient. Increasing the concentration of CO2 partially corrected the ethylene-mediated defects in NPQ and photosynthesis, indicating that changes in ethylene signaling affect stromal CO2 solubility. Increasing VDE expression in eto1-1 and ctr1-3 restored light-activated de-epoxidation and qE, reduced superoxide production and reduced photoinhibition. Restoring VDE activity significantly reversed the small growth phenotype of eto1-1 and ctr1-3 without altering ethylene production or ethylene responses. Our results demonstrate that ethylene increases ROS production and photosensitivity in response to high light and the associated reduced plant stature is partially reversed by increasing VDE activity.

Ethylene receptors in plants - why so much complexity?

Ethylene is a hormone involved in numerous aspects of growth, development, and responses to biotic and abiotic stresses in plants. Ethylene is perceived through its binding to endoplasmic reticulum-localized receptors that function as negative regulators of ethylene signaling in the absence of the hormone. In Arabidopsis thaliana, five structurally and functionally different ethylene receptors are present. These differ in their primary sequence, in the domains present, and in the type of kinase activity exhibited, which may suggest functional differences among the receptors. Whereas ethylene receptors functionally overlap to suppress ethylene signaling, certain other responses are controlled by specific receptors. In this review, I examine the nature of these receptor differences, how the evolution of the ethylene receptor gene family may provide insight into their differences, and how expression of receptors or their accessory proteins may underlie receptor-specific responses.

Appearance and elaboration of the ethylene receptor family during land plant evolution.

Ethylene is perceived following binding to endoplasmic reticulum-localized receptors, which in Arabidopsis thaliana, include ETR1, ERS1, EIN4, ETR2, and ERS2. These receptors fall into two subfamilies based on conservation of features within their histidine kinase domain. Subfamily 1 contains ETR1 and ERS1 whereas subfamily 2 contains EIN4, ETR2, and ERS2. Because ethylene receptors are found only in plants, this raises questions of when each receptor evolved. Here it is shown that subfamily 1 receptors encoded by a multigene family are present in all charophytes examined, these being most homologous to ETR1 based on their evolutionary relationship as well as containing histidine kinase and receiver domains. In charophytes and Physcomitrella patens, one or more gene family members contain the intron characteristic of subfamily 2 genes, indicating the first step in subfamily 2 receptor evolution. ERS1 homologs appear in basal angiosperm species after Amborella trichopoda and, in some early and basal angiosperm species and monocots in general, it is the only subfamily 1 receptor present. Distinct EIN4 and ETR2 homologs appear only in core eudicots and ERS2 homologs appear only in the Brassicaceae, suggesting it is the most recent receptor to evolve. These findings show that a subfamily 1 receptor had evolved and a subfamily 2 receptor had begun to evolve in plants prior to the colonization of land and only these two existed up to the appearance of the first basal angiosperm. The appearance of ERS2 in the Brassicaceae suggests ongoing evolution of the ethylene receptor family.

Phylogenetic analysis reveals dynamic evolution of the poly(A)-binding protein gene family in plants.

BACKGROUND: The poly(A)-binding protein (PABP) binds the poly(A) tail of eukaryotic mRNAs and functions to maintain the integrity of the mRNA while promoting protein synthesis through its interaction with eukaryotic translation initiation factor (eIF) 4G and eIF4B. PABP is encoded by a single gene in yeast and marine algae but during plant evolution the PABP gene family expanded substantially, underwent sequence divergence into three subclasses, and acquired tissue-specificity in gene family member expression. Although such changes suggest functional specialization, the size of the family and its sequence divergence have complicated an understanding of which gene family members may be foundational and which may represent more recent expansions of the family to meet the specific needs of speciation. Here, we examine the evolution of the plant PABP gene family to provide insight into these aspects of the family that may yield clues into the function of individual family members. RESULTS: The PABP gene family had expanded to two members by the appearance of fresh water algae and four members in non-vascular plants. In lycophytes, the first sequence divergence yielding a specific class member occurs. The earliest members of the gene family share greatest similarity to those modern members whose expression is confined to reproductive tissues, suggesting that supporting reproductive-associated gene expression is the most conserved function of this family. A family member sharing similarity to modern vegetative-associated members first appears in gymnosperms. Further elaboration of the reproductive-associated and vegetative-associated members occurred during the evolution of flowering plants. CONCLUSIONS: Expansion of the plant PABP gene family began prior to the colonization of land. By the evolution of lycophytes, the first class member whose expression is confined to reproductive tissues in higher plants had appeared. A second class member whose expression is vegetative-associated appeared in gymnosperms and all three modern classes had fully evolved by the appearance of the first known basal angiosperm. The size of each PABP class underwent further expansion during subsequent evolution, especially in the Brassicaceae, suggesting that the family is undergoing dynamic evolution.

Eukaryotic translation initiation factor eIFiso4G is required to regulate violaxanthin De-epoxidase expression in Arabidopsis.

The eukaryotic translation initiation factor (eIF) 4G is a scaffold protein that organizes the assembly of those initiation factors needed to recruit the 40 S ribosomal subunit to an mRNA. Plants, like many eukaryotes, express two eIF4G isoforms. eIFiso4G, one of the isoforms specific to plants, is unique among eukaryotic eIF4G proteins in that it is highly divergent and unusually small in size, raising the possibility of functional specialization. In this study, the role of eIFiso4G in plant growth was investigated using null mutants for the eIF4G isoforms in Arabidopsis. eIFiso4G loss of function mutants exhibited smaller cell, leaf, plant size, and biomass accumulation that correlated with its reduced photosynthetic activity, phenotypes not observed with the eIF4G loss of function mutant. Although no change in photorespiration or dark respiration was observed in the eIFiso4G loss of function mutant, a reduction in chlorophyll levels and an increase in the level of nonphotochemical quenching were observed. An increase in xanthophyll cycle activity and the generation of reactive oxygen species contributed to the qE and qI components of nonphotochemical quenching, respectively. An increase in the transcript and protein levels of violaxanthin de-epoxidase in the eIFiso4G loss of function mutant and an increase in its xanthophyll de-epoxidation state correlated with the higher qE associated with loss of eIFiso4G expression. These observations indicate that eIFiso4G expression is required to regulate violaxanthin de-epoxidase expression and to support photosynthetic activity.

The role of the poly(A) binding protein in the assembly of the Cap-binding complex during translation initiation in plants.

Translation initiation in eukaryotes requires the involvement of multiple initiation factors (eIFs) that facilitate the binding of the 40 S ribosomal subunit to an mRNA and assemble the 80 S ribosome at the correct initiation codon. eIF4F, composed of eIF4E, eIF4A, and eIF4G, binds to the 5'-cap structure of an mRNA and prepares an mRNA for recruitment of a 40 S subunit. eIF4B promotes the ATP-dependent RNA helicase activity of eIF4A and eIF4F needed to unwind secondary structure present in a 5'-leader that would otherwise impede scanning of the 40 S subunit during initiation. The poly(A) binding protein (PABP), which binds the poly(A) tail, interacts with eIF4G and eIF4B to promote circularization of an mRNA and stimulates translation by promoting 40 S subunit recruitment. Thus, these factors serve essential functions in the early steps of protein synthesis. Their assembly and function requires multiple interactions that are competitive in nature and determine the nature of interactions between the termini of an mRNA. In this review, the domain organization and partner protein interactions are presented for the factors in plants which share similarities with those in animals and yeast but differ in several important respects. The functional consequences of their interactions on factor activity are also discussed.

L-ascorbic Acid: a multifunctional molecule supporting plant growth and development.

L-Ascorbic acid (vitamin C) is as essential to plants as it is to animals. Ascorbic acid functions as a major redox buffer and as a cofactor for enzymes involved in regulating photosynthesis, hormone biosynthesis, and regenerating other antioxidants. Ascorbic acid regulates cell division and growth and is involved in signal transduction. In contrast to the single pathway responsible for ascorbic acid biosynthesis in animals, plants use multiple pathways to synthesize ascorbic acid, perhaps reflecting the importance of this molecule to plant health. Given the importance of ascorbic acid to human nutrition, several technologies have been developed to increase the ascorbic acid content of plants through the manipulation of biosynthetic or recycling pathways. This paper provides an overview of these approaches as well as the consequences that changes in ascorbic acid content have on plant growth and function. Discussed is the capacity of plants to tolerate changes in ascorbic acid content. The many functions that ascorbic acid serves in plants, however, will require highly targeted approaches to improve their nutritional quality without compromising their health.

Increasing vitamin C content in plant foods to improve their nutritional value-successes and challenges.

Vitamin C serves as a cofactor in the synthesis of collagen needed to support cardiovascular function, maintenance of cartilage, bones, and teeth, as well as being required in wound healing. Although vitamin C is essential, humans are one of the few mammalian species unable to synthesize the vitamin and must obtain it through dietary sources. Only low levels of the vitamin are required to prevent scurvy but subclinical vitamin C deficiency can cause less obvious symptoms such as cardiovascular impairment. Up to a third of the adult population in the U.S. obtains less than the recommended amount of vitamin C from dietary sources of which plant-based foods constitute the major source. Consequently, strategies to increase vitamin C content in plants have been developed over the last decade and include increasing its synthesis as well as its recycling, i.e., the reduction of the oxidized form of ascorbic acid that is produced in reactions back into its reduced form. Increasing vitamin C levels in plants, however, is not without consequences. This review provides an overview of the approaches used to increase vitamin C content in plants and the successes achieved. Also discussed are some of the potential limitations of increasing vitamin C and how these may be overcome.

The unique evolution of the programmed cell death 4 protein in plants.

BACKGROUND: The programmed cell death 4 (PDCD4) protein is induced in animals during apoptosis and functions to inhibit translation and tumor promoter-induced neoplastic transformation. PDCD4 is composed of two MA3 domains that share similarity with the single MA3 domain present in the eukaryotic translation initiation factor (eIF) 4G, which serves as a scaffold protein to assemble several initiation factors needed for the recruitment of the 40S ribosomal subunit to an mRNA. Although eIF4A is an ATP-dependent RNA helicase that binds the MA3 domain of eIF4G to promote translation initiation, binding of eIF4A to the MA3 domains of PDCD4 inhibits protein synthesis. Genes encoding PDCD4 are present in many lower eukaryotes and in plants, but PDCD4 in higher plants is unique in that it contains four MA3 domains and has been implicated in ethylene signaling and abiotic stress responses. Here, we examine the evolution of PDCD4 in plants. RESULTS: In older algal lineages, PDCD4 contains two MA3 domains similar to the homolog in animals. By the appearance of early land plants, however, PDCD4 is composed of four MA3 domains which likely is the result of a duplication of the two MA3 domain form of the protein. Evidence from fresh water algae, from which land plants evolved, suggests that the duplication event occurred prior to the colonization of land. PDCD4 in more recently evolved chlorophytes also contains four MA3 domains but this may have resulted from an independent duplication event. Expansion and divergence of the PDCD4 gene family occurred during land plant evolution with the appearance of a distinct gene member following the evolution of basal angiosperms. CONCLUSIONS: The appearance of a unique form of PDCD4 in plants correlates with the appearance of components of the ethylene signaling pathway, suggesting that it may represent the adaptation of an existing protein involved in programmed cell death to one that functions in abiotic stress responses through hormone signaling.

Eukaryotic initiation factor 4B and the poly(A)-binding protein bind eIF4G competitively.

The eukaryotic translation initiation factor (eIF) 4G functions as a scaffold protein that assembles components of the translation initiation complex required to recruit the 40S ribosomal subunit to an mRNA. Although many eukaryotes express two highly similar eIF4G isoforms, those in plants are highly divergent in size and sequence from one another and are referred to as eIF4G and eIFiso4G. Although the domain organization of eIFiso4G differs substantially from eIF4G orthologs in other species, the domain organization of plant eIF4G is largely unknown despite the fact that it is more similar in size and sequence to eIF4G of other eukaryotes. In this study, we show that eIF4G differs from eIFiso4G in that it contains two distinct interaction domains for the poly(A) binding protein (PABP) and eIF4B but is similar to eIFiso4G in having two eIF4A interaction domains. PABP and eIF4B bind the same N-terminal region of eIF4G as they do to a region C-proximal to the HEAT-1 domain in the middle domain of eIF4G, resulting in competitive binding between eIF4B and PABP to each site. eIF4G also differs from eIFiso4G in that no competitive binding was observed between PABP and eIF4A or between eIF4B and eIF4A to its HEAT-1-containing region. These results demonstrate that despite substantial differences in size, sequence, and domain organization, PABP and eIF4B bind to eIF4G and eIFiso4G competitively.

The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant.

BACKGROUND: Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1. RESULTS: The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs. CONCLUSIONS: These data represent the first genome-wide analysis of translation in a eukaryote defective in the large ribosomal subunit. RPL24 and eIF3h play similar but non-identical roles in eukaryotic translation. The data also shed light on the fine structure of the regulon of ribosomal protein mRNAs.

The role of L-ascorbic acid recycling in responding to environmental stress and in promoting plant growth.

L-Ascorbic acid (Asc) is the most abundant water-soluble antioxidant in plants. It serves as a cofactor for enzymes involved in photosynthesis, hormone biosynthesis, and the regeneration of antioxidants such as α-tocopherol. Once used, Asc can be recycled by several different mechanisms. The short-lived monodehydroascorbate (MDHA) radical, produced following Asc oxidation, can be recycled following reduction by ferredoxin or monodehydroascorbate reductase (MDAR). MDHA can also undergo disproportionation into dehydroascorbate (DHA) and Asc. DHA can be recycled into Asc by dehydroascorbate reductase (DHAR) before it undergoes irrevocable hydrolysis. Through its recycling, Asc content and its redox state are maintained, which is critical under conditions of high demand, for example during high light or other stress conditions that increase reactive oxygen species (ROS) production. This review provides an overview of research in the last decade revealing the role that Asc recycling plays during germination, growth, and reproduction, as well as in response to environmental stress. These findings highlight the importance of DHAR- and MDAR-mediated mechanisms of Asc recycling in maintaining ROS at non-damaging levels while modulating ROS signalling function.

Metabolite profiling of Arabidopsis inoculated with Alternaria brassicicola reveals that ascorbate reduces disease severity.

The interaction between the pathogenic ascomycete Alternaria brassicicola and Arabidopsis was investigated by metabolite profiling. The effect of A. brassicicola challenge on metabolite levels was substantial, with nearly 50% of detected compounds undergoing significant changes. Mutations blocking ethylene, jasmonic acid, or ethylene signaling had little effect on metabolite levels. The effects of altering levels of some metabolites were tested by exogenous application during A. brassicicola inoculation. Gamma amino-butyric acid (GABA) or xylitol promoted, while trehalose and ascorbate inhibited, disease severity. GABA promoted, and ascorbate strongly inhibited, fungal growth in culture. Arabidopsis vtc1 and vtc2 mutants, that have low levels of ascorbate, were more susceptible to A. brassicicola. Ascorbate levels declined following A. brassicicola inoculation while levels of dehydroascorbate increased, resulting in a shift of the redox balance between these compounds in the direction of oxidation. These results demonstrate that ascorbate is an important component of resistance to this pathogen.

Violaxanthin de-epoxidase is rate-limiting for non-photochemical quenching under subsaturating light or during chilling in Arabidopsis.

In response to conditions of excess light energy, plants induce non-photochemical quenching (NPQ) as a protective mechanism to prevent over reduction of photosystem II and the generation of reactive oxygen species (ROS). The xanthophyll cycle, which contributes significantly to reversible NPQ to thermally dissipate excess absorbed light energy, involves de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin in response to excess light energy. The activation of violaxanthin de-epoxidase (VDE), which catalyzes the de-epoxidation reaction, requires the generation of a light-induced, transthylakoid pH gradient. In this work, we overexpressed or repressed the expression of VDE in Arabidopsis (Arabidopsis thaliana) to examine whether VDE is rate-limiting for the induction of NPQ. Increasing VDE expression increased the de-epoxidation state of xanthophyll pigments, the rate of NPQ induction, and the level of NPQ achieved under subsaturating light. In saturating light, however, overexpression of VDE did not increase the xanthophyll pigment de-epoxidation state, the level of NPQ achieved following its initial induction, or substantially improve tolerance to high light. Only under chilling, which reduces VDE activity, did an increase in VDE expression provide slightly greater phototolerance. Repression of VDE expression impaired violaxanthin de-epoxidation, reduced the generation of NPQ, and lowered the level of NPQ achieved while increasing photosensitivity. These results demonstrate that the endogenous level of VDE is rate-limiting for NPQ in Arabidopsis under subsaturating but not saturating light and can become rate-limiting under chilling conditions. These results also show that increasing VDE expression confers greater phototolerance mainly under conditions which limit endogenous VDE activity.

Induction of monozygotic twinning by ascorbic acid in tobacco.

Embryo development in plants initiates following the transverse division of a zygote into an apical, proembryo cell and a basal cell that gives rise to the suspensor. Although mutants affected in embryo development through changes in cell division have been described, little is known about the control of the first zygotic division that gives rise to the proembryo. Ascorbic acid (Asc) promotes cell division by inducing G(1) to S progression but its role in embryo development has not been examined. In this study, we show that the level of dehydroascorbate reductase (DHAR) expression, which recycles Asc and regulates Asc pool size, affects the rate of monozygotic twinning and polycotyly. DHAR-induced twinning resulted from altered cell polarity and longitudinal instead of transverse cell division that generated embryos of equal size. Direct injection of Asc into ovaries phenocopied DHAR-induced twinning. Twinning induced by Asc was developmentally limited to the first two days after pollination whereas polycotyly was induced when the level of Asc was elevated just prior to cotyledon initiation. This work describes the first example of gene-directed monozygotic twinning and shows that Asc regulates cell polarity during embryo development.

Analysis of the functional conservation of ethylene receptors between maize and Arabidopsis.

Ethylene, a regulator of plant growth and development, is perceived by specific receptors that act as negative regulators of the ethylene response. Five ethylene receptors, i.e., ETR1, ERS1, EIN4, ETR2, and ERS2, are present in Arabidopsis and dominant negative mutants of each that confer ethylene insensitivity have been reported. In contrast, maize contains just two types of ethylene receptors: ZmERS1, encoded by ZmERS1a and ZmERS1b, and ZmETR2, encoded by ZmETR2a and ZmETR2b. In this study, we introduced a Cys to Tyr mutation in the transmembrane domain of ZmERS1b and ZmETR2b that is present in the etr1-1 dominant negative mutant and expressed each protein in Arabidopsis. Mutant Zmers1b and Zmetr2b receptors conferred ethylene insensitivity and Arabidopsis expressing Zmers1b or Zmetr2b were larger and exhibited a delay in leaf senescence characteristic of ethylene insensitive Arabidopsis mutants. Zmers1b and Zmetr2b were dominant and functioned equally well in a hemizygous or homozygous state. Expression of the Zmers1b N-terminal transmembrane domain was sufficient to exert dominance over endogenous Arabidopsis ethylene receptors whereas the Zmetr2b N-terminal domain failed to do so. Neither Zmers1b nor Zmetr2b functioned in the absence of subfamily 1 ethylene receptors, i.e., ETR1 and ERS1. These results suggest that Cys65 in maize ZmERS1b and ZmETR2b plays the same role that it does in Arabidopsis receptors. Moreover, the results demonstrate that the mutant maize ethylene receptors are functionally dependent on subfamily 1 ethylene receptors in Arabidopsis, indicating substantial functional conservation between maize and Arabidopsis ethylene receptors despite their sequence divergence.