Chloroplasts are the metabolically active, semi-autonomous organelles found in plants, algae and cyanobacteria. Their main function is to carry out the photosynthesis process involving a conversion of light energy into the energy of chemical bonds used for the synthesis of organic compounds. The Chloroplasts’ proteome consists of several thousand proteins that, besides photosynthesis, participate in the biosynthesis of fatty acids, amino acids, hormones, vitamins, nucleotides and secondary metabolites. Most of the chloroplast proteins are nuclear-encoded. During the course of evolution, many genes of the ancestral chloroplasts have been transferred from the chloroplast genome into the cell nucleus. However, these proteins which are essential for the photosynthesis have been retained in the chloroplast genome. This review aims to provide a relatively comprehensive summary of the knowledge in the field of the chloroplast genome arrangement and the chloroplast genes expression process based on a widely used model in plant genetic research, namely Arabidopsis thaliana.

New players in plant signaling are described in detail in this review: extracellular ATP (eATP) and uncommon nucleotides such as dinucleoside polyphosphates (Np_nN’s), adenosine 5′-phosphoramidate (NH₂-pA), and extracellular NAD⁺ and NADP⁺ (eNAD(P)⁺). Recent molecular, physiological, and biochemical evidence implicating concurrently the signaling role of eATP, Np_nN’s, and NH₂-pA in plant biology and the mechanistic events in which they are involved are discussed. Numerous studies have shown that they are often universal signaling messengers, which trigger a signaling cascade in similar reactions and processes among different kingdoms. We also present here, not described elsewhere, a working model of the Np_nN’ and NH₂-pA signaling network in a plant cell where these nucleotides trigger induction of the phenylpropanoid and the isochorismic acid pathways yielding metabolites protecting the plant against various types of stresses. Through these signals, the plant responds to environmental stimuli by intensifying the production of various compounds, such as anthocyanins, lignin, stilbenes, and salicylic acid. Still, more research needs to be performed to identify signaling networks that involve uncommon nucleotides, followed by omic experiments to define network elements and processes that are controlled by these signals.

Plants adjust their architecture to a constantly changing environment, requiring adaptation of differential growth. Despite their importance, molecular switches, which define growth transitions, are largely unknown. Apical hook development in dark grown Arabidopsis thaliana (A. thaliana) seedlings serves as a suitable model for differential growth transition in plants. Here, we show that the phytohormone auxin counteracts the light-induced growth transition during apical hook opening. We, subsequently, identified genes which are inversely regulated by light and auxin. We used in silico analysis of the regulatory elements in this set of genes and subsequently used natural variation in gene expression to uncover correlations between underlying transcription factors and the in silico predicted target genes. This approach uncovered that MADS box transcription factor AGAMOUS-LIKE 8 (AGL8)/FRUITFULL (FUL) modulates apical hook opening. Our data shows that transient FUL expression represses the expression of growth stimulating genes during early phases of apical hook development and therewith guards the transition to growth promotion for apical hook opening. Here, we propose a role for FUL in setting tissue identity, thereby regulating differential growth during apical hook development.

Dinucleoside polyphosphates (Np_nNs) are considered novel signalling molecules involved in the induction of plant defence mechanisms. However, Np_nN signal recognition and transduction are still enigmatic. Therefore, the aim of our research was the identification of the Np_nN receptor and signal transduction pathways evoked by these nucleotides. Earlier, we proved that purine and pyrimidine Np_nNs differentially affect the phenylpropanoid pathway in Vitis vinifera suspension-cultured cells. Here, we report, for the first time, that both diadenosine tetraphosphate (Ap₄A) and dicytidine tetraphosphate (Cp₄C)-induced stomatal closure in Arabidopsis thaliana. Moreover, we showed that plasma membrane purinoreceptor P2K1/DORN1 (does not respond to nucleotide 1) is essential for Ap₄A-induced stomata movements but not for Cp₄C. Wild-type Col-0 and the dorn1-3 A. thaliana knockout mutant were used. Examination of the leaf epidermis dorn1-3 mutant provided evidence that P2K1/DORN1 is a part of the signal transduction pathway in stomatal closure evoked by extracellular Ap₄A but not by Cp₄C. Reactive oxygen species (ROS) are involved in signal transduction caused by Ap₄A and Cp₄C, leading to stomatal closure. Ap₄A induced and Cp₄C suppressed the transcriptional response in wild-type plants. Moreover, in dorn1-3 leaves, the effect of Ap₄A on gene expression was impaired. The interaction between P2K1/DORN1 and Ap₄A leads to changes in the transcription of signalling hubs in signal transduction pathways.

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (Np_nN’s) and adenosine 5′-phosphoramidate (NH₂-pA) belonging to nucleoside 5′-phosphoramidates (NH₂-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that Np_nN’s induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH₂-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH₂-pA, NH₂-pG) and pyrimidine (NH₂-pU, NH₂-pC) nucleoside 5′-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH₂-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH₂-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5′-phosphoramidates could be considered as new signaling molecules.

Summary: Dinucleoside polyphosphates (Np _n Ns) are considered novel signalling molecules involved in the induction of plant defence mechanisms. However, the Np _n Ns signal recognition and transduction are still enigmatic. Here we report, for the first time, that diadenosine tetraphosphate (Ap ₄ A) is recognized by the Arabidopsis thaliana purinoreceptor P2K1/DORN1 (Does Not Respond to Nucleotides 1) and causes stomatal closure. Extracellular Ap ₄ A- and dicytidine tetraphosphate (Cp ₄ C)-induced stomatal closure was observed using a microscope. Reactive oxygen species (ROS) accumulation was determined by staining with nitroblue tetrazolium (NBT) and 3,3ʹ-diaminobenzidine tetrahydrochloride (DAB). Transcriptional changes were determined by quantitative real-time PCR. Wild-type Col-0 and the dorn1-3 A. thaliana knockout mutant were used. Examination of the leaf epidermis dorn1-3 mutant provided evidence that P2K1/DORN1 recognizes extracellular Ap ₄ A but not Cp ₄ C. ROS are involved in signal transduction caused by Ap ₄ A and Cp ₄ C, leading to stomatal closure. Ap ₄ A induced and Cp ₄ C suppressed the transcriptional response in wild-type plants. Moreover, in dorn1-3 leaves, the effect of Ap ₄ A on gene expression was impaired. Our research demonstrated, for the first time, that P2K1/DORN1 is a plant purinoreceptor for Ap ₄ A. This interaction leads to changes in the transcription of signalling hubs in signal transduction pathways.