Divergent regulation of the HEMA gene family encoding glutamyl-tRNA reductase in Arabidopsis thaliana: expression of HEMA2 is regulated by sugars,but is independent of light and plastid signalling

The synthesis of 5-aminolevulinic acid (ALA) is a key regulatory step for the production of hemes and chlorophyll via the tetrapyrrole synthesis pathway. The first enzyme committed to ALA synthesis is glutamyl-tRNA reductase encoded in Arabidopsis by a small family of nuclear-encoded HEMA genes. To better understand the regulation of the tetrapyrrole synthesis pathway we have made a detailed study of HEMA2 expression with transgenic Arabidopsis thaliana L. Col. plants carrying chimeric HEMA2 promoter:gusA fusion constructs. Our results show that the HEMA2 promoter directs expression predominantly to roots and flowers, but that HEMA2 is also expressed at low levels in photosynthetic tissues. Deletion analysis of the HEMA2 promoter indicates that a ca. 850 bp fragment immediately upstream of the HEMA2 coding region is sufficient to drive regulated gusA expression. In contrast to HEMA1, HEMA2 is not up-regulated by red, far-red, blue, UV or white light. In addition, elimination of a promotive plastid signal by Norflurazon-induced photobleaching of plastids had no effect on HEMA2 expression while being required for normal white-light induction of HEMA1. HEMA2 expression in the cotyledons is inhibited by the presence of sucrose or glucose, but not fructose, and this response is light-independent. HEMA1 expression in cotyledons is also inhibited by sugars, but in a strictly light-dependent manner. The roles of HEMA1 and HEMA2 in meeting cellular tetrapyrrole requirements are discussed.     

Overexpression of HEMA1 encoding glutamyl-tRNA reductase

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50-90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.     

Concurrent interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark- and light-grown Arabidopsis plants

The regulation of tetrapyrrole biosynthesis in higher plants has been attributed to metabolic feedback inhibition of Glu tRNA reductase by heme. Recently, another negative regulator of tetrapyrrole biosynthesis has been discovered, the FLU protein. During an extensive second site screen of mutagenized flu seedlings a suppressor of flu, ulf3, was identified that is allelic to hy1 and encodes a heme oxygenase. Increased levels of heme in the hy1 mutant have been implicated with inhibiting Glu tRNA reductase and suppressing the synthesis of delta-aminolevulinic acid (ALA) and Pchlide accumulation. When combined with hy1 or ulf3 upregulation of ALA synthesis and overaccumulation of protochlorophyllide in the flu mutants were severely suppressed supporting the notion that heme antagonizes the effect of the flu mutation by inhibiting Glu tRNA reductase independently of FLU. The coiled-coil domain at the C-terminal end of Glu tRNA reductase interacts with FLU, whereas the N-terminal site of Glu tRNA reductase that is necessary for the inhibition of the enzyme by heme is not required for this interaction. The interaction with FLU is specific for the Glu tRNA reductase encoded by HEMA1 that is expressed in photosynthetically active tissues. FLU seems to be part of a second regulatory circuit that controls chlorophyll biosynthesis by interacting directly with Glu tRNA reductase not only in etiolated seedlings but also in light-adapted green plants.     

Selective inhibition of HEMA gene expression by photooxidation in Arabidopsis thaliana.

Norflurazon (NF), a photobleaching herbicide, inhibits carotenoid biosynthesis. Lack of carotenoid pigments leads to photooxidative damage of chloroplasts. In this study of Arabidopsis thaliana we demonstrate that NF-treated photobleached plants are still able to make 5-aminolevulinic acid (ALA) the first precursor of porphyrins and tetrapyrroles. ALA is formed in the tRNA-dependent two-step C5-pathway in the chloroplast of plants. The expression of glutamyl-tRNA reductase (GluTR), the first enzyme in the pathway, was severely inhibited by NF, while treatment with this compound did not significantly reduce the levels of the other enzyme, glutamate-l-semialdehyde aminomutase, or of tRNA(Glu), the initial metabolite of the pathway. Extracts of these plants retained the capacity, albeit reduced, to convert exogenously added glutamate to ALA. Thus, the much-reduced level of ALA formation in photobleached plants is due to selective inhibition of GluTR expression.     

Light-signalling pathways leading to the co-ordinated expression of HEMA1 and Lhcb during chloroplast development in Arabidopsis thaliana

During de-etiolation, the co-ordinated synthesis of chlorophyll and the chlorophyll a/b-binding proteins is critical to the development of functional light-harvesting complexes. To understand how this co-ordination is achieved, we have made a detailed study of the light-regulated signalling pathways mediating the expression of the HEMA1 and Lhcb genes encoding glutamyl-tRNA reductase, the first committed enzyme of 5-aminolaevulinic acid formation, and chlorophyll a/b-binding proteins, respectively. To do this, we have screened 7 photoreceptor and 12 light-signalling mutants of Arabidopsis thaliana L. for induction of HEMA1 and Lhcb expression in continuous red, far-red and blue light and following a red pulse. We have categorised these mutants into two groups. The phyA, phyB, phyAphyB, cry1, cry2, cop1, det1, poc1, eid1, and far1 mutations lead to diverse effects on the light regulation of HEMA1, but affect Lhcb expression to a similar degree. The hy1, hy2, hy5, fin219, fhy1, fhy3, spa1, ndpk2, and pat1 mutants also affect light regulation of both HEMA1 and Lhcb expression, but with differences in the relative magnitude of the two responses. The fhy1 and fhy3 mutants show the most significant differences in light regulation between the two genes, with both showing a strong inhibition of HEMA1 expression under continuous red light. These results demonstrate that co-ordinated regulation of HEMA1 and Lhcb is largely achieved through parallel light regulation mediated by shared phytochrome- and cryptochrome-signalling pathways. However, glutamyl-tRNA reductase is also required for the synthesis of other tetrapyrroles and this dual role may account for the observed differences in these light-signalling pathways.     

Glutamate 1-semialdehyde aminotransferase is connected to GluTR by GluTR-binding protein and contributes to the rate-limiting step of 5-aminolevulinic acid synthesis

Tetrapyrroles play fundamental roles in crucial processes including photosynthesis, respiration, and catalysis. In plants, 5-aminolevulinic acid (ALA) is the common precursor of tetrapyrroles. ALA is synthesized from activated glutamate by the enzymes glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAAT). ALA synthesis is recognized as the rate-limiting step in this pathway. We aimed to explore the contribution of GSAAT to the control of ALA synthesis and the formation of a protein complex with GluTR. In Arabidopsis thaliana, two genes encode GSAAT isoforms: GSA1 and GSA2. A comparison of two GSA knockout mutants with the wild-type revealed the correlation of reduced GSAAT activity and ALA-synthesizing capacity in leaves with lower chlorophyll content. Growth and green pigmentation were more severely impaired in gsa2 than in gsa1, indicating the predominant role of GSAAT2 in ALA synthesis. Interestingly, GluTR accumulated to higher levels in gsa2 than in the wild-type and was mainly associated with the plastid membrane. We propose that the GSAAT content modulates the amount of soluble GluTR available for ALA synthesis. Several different biochemical approaches revealed the GSAAT–GluTR interaction through the assistance of GluTR-binding protein (GBP). A modeled structure of the tripartite protein complex indicated that GBP mediates the stable association of GluTR and GSAAT for adequate ALA synthesis.

A mechanism is described that maintains adequate 5-aminolevulinic acid activity via a tripartite protein complex, representing the rate-limiting step in the synthesis of vital tetrapyrrole pigments.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.)

Two putative glycosyltransferases in Arabidopsis thaliana, designated reduced residual arabinose-1 and -2 (RRA1 and RRA2), are characterized at the molecular level. Both genes are classified in CAZy GT-family-77 and are phylogenetically related to putative glycosyltranferases of Chlamydomonas reinhardtii. The expression pattern of the two genes was analyzed by semi-quantitative RT-PCR using mRNA extracted from various organs of bolting Arabidopsis thaliana plants. In addition, promoter::gusA analysis of transgenic Arabidopsis thaliana containing a fusion between either the RRA-1 or -2 promoter fragment and the gusA reporter gene showed that whereas the RRA1 promoter was primarily active in the apical meristem, the expression pattern of the RRA2 promoter was more diverse but also highly active in the meristematic region. In addition, T-DNA mutant insertion lines of both RRA-1 and -2, were identified and characterized at the molecular and biochemical level. Monosaccharide compositional analyses of cell wall material isolated from the meristematic region showed a ca. 20% reduction in the arabinose content in the insoluble/undigested cell wall residue after enzymatic removal of xyloglucan and pectic polysaccharides. These data indicate that both RRA-1 and -2 play a role in the arabinosylation of cell wall component(s).     

Feedback Inhibition of Chlorophyll Synthesis in the Phytochrome Chromophore-Deficient aurea and yellow-green-2 Mutants of Tomato

The aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) are unable to synthesize the linear tetrapyrrole chromophore of phytochrome, resulting in plants with a yellow-green phenotype. To understand the basis of this phenotype, we investigated the consequences of the au and yg-2 mutations on tetrapyrrole metabolism. Dark-grown seedlings of both mutants have reduced levels of protochlorophyllide (Pchlide) due to an inhibition of Pchlide synthesis. Feeding experiments with the tetrapyrrole precursor 5-aminolevulinic acid (ALA) demonstrate that the pathway between ALA and Pchlide is intact in au and yg-2 and suggest that the reduction in Pchlide is a result of the inhibition of ALA synthesis. This inhibition was independent of any deficiency in seed phytochrome, and experiments using an iron chelator to block heme synthesis demonstrated that both mutations inhibited the degradation of the physiologically active heme pool, suggesting that the reduction in Pchlide synthesis is a consequence of feedback inhibition by heme. We discuss the significance of these results in understanding the chlorophyll-deficient phenotype of the au and yg-2 mutants.     

Green or red: what stops the traffic in the tetrapyrrole pathway?

Regulation of tetrapyrrole biosynthesis is crucial to plant metabolism. The two pivotal control points are formation of the initial precursor, 5-aminolaevulinic acid (ALA), and the metal-ion insertion step: chelation of Fe(2+) into protoporphyrin IX leads to haem and phytochromobilin, whereas insertion of Mg(2+) is the first step to chlorophyll. Recent studies with mutants and transgenic plants have demonstrated that perturbation of the branch point affects ALA formation. Moreover, one of the signals that controls the expression of genes for nuclear-encoded chloroplast proteins has been shown to be Mg-protoporphyrin-IX. Here, we discuss the regulation of branch-point flux and the relative contributions of the haem and chlorophyll branches to the regulation of ALA synthesis and thus to flow through the tetrapyrrole pathway.