Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD+ to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism.

Spike lavender (Lavandula latifolia) is an aromatic shrub cultivated worldwide for the production of essential oils. The major constituents of these oils are monoterpenes, which are obtained from isopentenyl diphosphate and dimethylallyl diphosphate precursors through the plastidial methylerythritol phosphate (MEP) pathway and/or the cytosolic mevalonate pathway. 1-Deoxy-d-xylulose-5-P synthase (DXS) catalyzes the first step of the MEP pathway. A cDNA coding for the Arabidopsis (Arabidopsis thaliana) DXS was constitutively expressed in spike lavender. Gas chromatography/mass spectrometry analyses revealed that transgenic plants accumulated significantly more essential oils compared to controls (from 101.5% to 359.0% and from 12.2% to 74.1% yield increase compared to controls in leaves and flowers, respectively). T0 transgenic plants were grown for 2 years, self-pollinated, and the T1 seeds obtained. The inheritance of the DXS transgene was studied in the T1 generation. The increased essential oil phenotype observed in the transgenic T0 plants was maintained in the progeny that inherited the DXS transgene. Total chlorophyll and carotenoid content in DXS progenies that inherited the transgene depended on the analyzed plant, showing either no variation or a significant decrease in respect to their counterparts without the transgene. Transgenic plants had a visual phenotype similar to untransformed plants (controls) in terms of morphology, growth habit, flowering, and seed germination. Our results demonstrate that the MEP pathway contributes to essential oil production in spike lavender. They also demonstrate that the DXS enzyme plays a crucial role in monoterpene precursor biosynthesis and, thus, in essential oil production in spike lavender. In addition, our results provide a strategy to increase the essential oil production in spike lavender by metabolic engineering of the MEP pathway without apparent detrimental effects on plant development and fitness.

Plant metabolism is highly coordinated with development. However, an understanding of the whole picture of metabolism and its interactions with plant development is scarce. In this work, we show that the deficiency in the plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) leads to male sterility in Arabidopsis (Arabidopsis thaliana). Pollen from homozygous gapcp double mutant plants (gapcp1gapcp2) displayed shrunken and collapsed forms and were unable to germinate when cultured in vitro. The pollen alterations observed in gapcp1gapcp2 were attributed to a disorganized tapetum layer. Accordingly, the expression of several of the genes involved in tapetum development was down-regulated in gapcp1gapcp2. The fertility of gapcp1gapcp2 was rescued by transforming this mutant with a construct carrying the GAPCp1 cDNA under the control of its native promoter (pGAPCp1::GAPCp1c). However, the GAPCp1 or GAPCp2 cDNA under the control of the 35S promoter (p35S::GAPCp), which is poorly expressed in the tapetum, did not complement the mutant fertility. Mutant GAPCp isoforms deficient in the catalytic activity of the enzyme were unable to complement the sterile phenotype of gapcp1gapcp2, thus confirming that both the expression and catalytic activity of GAPCp in anthers are necessary for mature pollen development. A metabolomic study in flower buds indicated that the most important difference between the sterile (gapcp1gapcp2, gapcp1gapcp2-p35S::GAPCp) and the fertile (wild-type plants, gapcp1gapcp2-pGAPCp1::GAPCp1c) lines was the increase in the signaling molecule trehalose. This work corroborates the importance of plastidial glycolysis in plant metabolism and provides evidence for the crucial role of GAPCps in pollen development. It additionally brings new insights into the complex interactions between metabolism and development.

In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate (3-PGA) in glycolysis, but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases we found three genes that encode putative PGKs. PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and non-photosynthetic cells. PGK3 was ubiquitously expressed in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the photosynthetic isoform, while PGK2 and PGK3 were the plastidial and cytosolic glycolytic isoforms, respectively. The pgk1.1 knock-down mutant displayed reduced growth, lower photosynthetic capacity and starch content. The pgk3.2 knock-out mutant was characterized by a reduced growth, but a higher starch levels than the wild-type. The pgk1.1 pgk3.2 double mutant was bigger than pgk3.2, and displayed an intermediate phenotype between the two single mutants in all measured biochemical and physiological parameters. Expression studies in PGK mutants showed that PGK1 and PGK3 were down-regulated in pgk3.2 and pgk1.1, respectively. These results indicate that the down-regulation of photosynthetic activity could be a plant strategy when glycolysis is impaired to achieve metabolic adjustment and optimize growth. The double mutants of PGK3 and the triose-phosphate transporter (pgk3.2 tpt3) displayed a drastic growth phenotype, but were viable. This implies that other enzymes or non-specific chloroplast transporters could provide 3-PGA to the cytosol. Our results highlight both the complexity and the plasticity of the plant primary metabolic network.

This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.