Cytosolic invertases contribute to cellulose biosynthesis and influence carbon partitioning in seedlings of Arabidopsis thaliana

In plants, UDP‐glucose is the direct precursor for cellulose biosynthesis, and can be converted into other NDP‐sugars required for the biosynthesis of wall matrix polysaccharides. UDP‐glucose is generated from sucrose by two distinct metabolic pathways. The first pathway is the direct conversion of sucrose to UDP‐glucose and fructose by sucrose synthase. The second pathway involves sucrose hydrolysis by cytosolic invertase (CINV), conversion of glucose to glucose‐6‐phosphate and glucose‐1‐phosphate, and UDP‐glucose generation by UDP‐glucose pyrophosphorylase (UGP). Previously, Barratt et al. (Proc. Natl Acad. Sci. USA, 106, 2009 and 13124) have found that an Arabidopsis double mutant lacking CINV1 and CINV2 displayed drastically reduced growth. Whether this reduced growth is due to deficient cell wall production caused by limited UDP‐glucose supply, pleiotropic effects, or both, remained unresolved. Here, we present results indicating that the CINV/UGP pathway contributes to anisotropic growth and cellulose biosynthesis in Arabidopsis. Biochemical and imaging data demonstrate that cinv1 cinv2 seedlings are deficient in UDP‐glucose production, exhibit abnormal cellulose biosynthesis and microtubule properties, and have altered cellulose organization without substantial changes to matrix polysaccharide composition, suggesting that the CINV/UGP pathway is a key metabolic route to UDP‐glucose synthesis in Arabidopsis. Furthermore, differential responses of cinv1 cinv2 seedlings to exogenous sugar supplementation support a function of CINVs in influencing carbon partitioning in Arabidopsis. From these data and those of previous studies, we conclude that CINVs serve central roles in cellulose biosynthesis and carbon allocation in Arabidopsis.     

Diurnal changes in concerted plant protein phosphorylation and acetylation in Arabidopsis organs and seedlings

Protein phosphorylation and acetylation are the two most abundant post‐translational modifications (PTMs) that regulate protein functions in eukaryotes. In plants, these PTMs have been investigated individually; however, their co‐occurrence and dynamics on proteins is currently unknown. Using Arabidopsis thaliana, we quantified changes in protein phosphorylation, acetylation and protein abundance in leaf rosettes, roots, flowers, siliques and seedlings at the end of day (ED) and at the end of night (EN). This identified 2549 phosphorylated and 909 acetylated proteins, of which 1724 phosphorylated and 536 acetylated proteins were also quantified for changes in PTM abundance between ED and EN. Using a sequential dual‐PTM workflow, we identified significant PTM changes and intersections in these organs and plant developmental stages. In particular, cellular process‐, pathway‐ and protein‐level analyses reveal that the phosphoproteome and acetylome predominantly intersect at the pathway‐ and cellular process‐level at ED versus EN. We found 134 proteins involved in core plant cell processes, such as light harvesting and photosynthesis, translation, metabolism and cellular transport, that were both phosphorylated and acetylated. Our results establish connections between PTM motifs, PTM catalyzing enzymes and putative substrate networks. We also identified PTM motifs for further characterization of the regulatory mechanisms that control cellular processes during the diurnal cycle in different Arabidopsis organs and seedlings. The sequential dual‐PTM analysis expands our understanding of diurnal plant cell regulation by PTMs and provides a useful resource for future analyses, while emphasizing the importance of analyzing multiple PTMs simultaneously to elucidate when, where and how they are involved in plant cell regulation.     

Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes

Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS‐ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS‐ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi‐in vitro, whereas normal PT growth was shown in its sense control in vitro/semi‐in vitro. PT growth was impaired in a manner dependent on the dose of AS‐ODNs against both ANX1 and ANX2 above 10 μm . The treatment with AS‐ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS‐ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS‐ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS‐ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.     

Identification and characterization of a plastidial phosphatidylglycerophosphate phosphatase in Arabidopsis thaliana

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1‐2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1‐1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1‐1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation.     

Cadmium‐induced and trans‐generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana

Trans‐generational adaptation is important to respond rapidly to environmental challenges and increase overall plant fitness. Besides well‐known mechanisms such as epigenetic modifications, vertically transmitted endophytic bacteria might contribute to this process. The cultivable and total endophytic communities of several generations of Arabidopsis thaliana seeds harvested from plants exposed to cadmium (Cd) or not exposed were investigated. The diversity and richness of the seed endophytic community decreased with an increasing number of generations. Aeromicrobium and Pseudonocardia were identified as indicator species in seeds from Cd‐exposed plants, while Rhizobium was abundantly present in both seed types. Remarkably, Rhizobium was the only genus that was consistently detected in seeds of all generations, which suggests that the phenotypic characteristics were more important as selection criteria for which bacteria are transferred to the next plant generation than the actual genera. Production of IAA was an important trait for endophytes from both seed types, while ACC deaminase activity and Cd tolerance were mainly associated with seed endophytes from Cd‐exposed plants. Understanding how different factors influence the seed endophytic community can help us to improve seed quality and plant growth through different biotechnological applications.     

Surface wax esters contribute to drought tolerance in Arabidopsis

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.     

Functional characterization of Arabidopsis phototropin 1 in the hypocotyl apex

Phototropin (phot1) is a blue light‐activated plasma membrane‐associated kinase that acts as the principal photoreceptor for shoot phototropism in Arabidopsis in conjunction with the signalling component Non‐Phototropic Hypocotyl 3 (NPH3). PHOT1 is uniformly expressed throughout the Arabidopsis hypocotyl, yet decapitation experiments have localized the site of light perception to the upper hypocotyl. This prompted us to investigate in more detail the functional role of the hypocotyl apex, and the regions surrounding it, in establishing phototropism. We used a non‐invasive approach where PHOT1–GFP (P1–GFP) expression was targeted to the hypocotyl apex of the phot‐deficient mutant using the promoters of CUP‐SHAPED COTYLEDON 3 (CUC3) and AINTEGUMENTA (ANT). Expression of CUC3::P1–GFP was clearly visible at the hypocotyl apex, with weaker expression in the cotyledons, whereas ANT::P1–GFP was specifically targeted to the developing leaves. Both lines showed impaired curvature to 0.005 μmol m^?2 sec^?1 unilateral blue light, indicating that regions below the apical meristem are necessary for phototropism. Curvature was however apparent at higher fluence rates. Moreover, CUC3::P1–GFP partially or fully complemented petiole positioning, leaf flattening and chloroplast accumulation, but not stomatal opening. Yet, tissue analysis of NPH3 de‐phosphorylation showed that CUC3::P1–GFP and ANT::P1–GFP mis‐express very low levels of phot1 that likely account for this responsiveness. Our spatial targeting approach therefore excludes the hypocotyl apex as the site for light perception for phototropism and shows that phot1‐mediated NPH3 de‐phosphorylation is tissue autonomous and occurs more prominently in the basal hypocotyl.     

MyROOT: a method and software for the semiautomatic measurement of primary root length in Arabidopsis seedlings

Root analysis is essential for both academic and agricultural research. Despite the great advances in root phenotyping and imaging, calculating root length is still performed manually and involves considerable amounts of labor and time. To overcome these limitations, we developed MyROOT, a software for the semiautomatic quantification of root growth of seedlings growing directly on agar plates. Our method automatically determines the scale from the image of the plate, and subsequently measures the root length of the individual plants. To this aim, MyROOT combines a bottom‐up root tracking approach with a hypocotyl detection algorithm. At the same time as providing accurate root measurements, MyROOT also significantly minimizes the user intervention required during the process. Using Arabidopsis, we tested MyROOT with seedlings from different growth stages and experimental conditions. When comparing the data obtained from this software with that of manual root measurements, we found a high correlation between both methods (R² = 0.997). When compared with previous developed software with similar features (BRAT and EZ‐Rhizo), MyROOT offered an improved accuracy for root length measurements. Therefore, MyROOT will be of great use to the plant science community by permitting high‐throughput root length measurements while saving both labor and time.     

Gene family evolution in green plants with emphasis on the origination and evolution of Arabidopsis thaliana genes

Gene family size variation is an important mechanism that shapes the natural variation for adaptation in various species. Despite its importance, the pattern of gene family size variation in green plants is still not well understood. In particular, the evolutionary pattern of genes and gene families remains unknown in the model plant Arabidopsis thaliana in the context of green plants. In this study, eight representative genomes of green plants are sampled to study gene family evolution and characterize the origination of A. thaliana genes, respectively. Four important insights gained are that: (i) the rate of gene gains and losses is about 0.001359 per gene every million years, similar to the rate in yeast, Drosophila, and mammals; (ii) some gene families evolved rapidly with extreme expansions or contractions, and 2745 gene families present in all the eight species represent the ‘core’ proteome of green plants; (iii) 70% of A. thaliana genes could be traced back to 450 million years ago; and (iv) intriguingly, A. thaliana genes with early origination are under stronger purifying selection and more conserved. In summary, the present study provides genome‐wide insights into evolutionary history and mechanisms of genes and gene families in green plants and especially in A. thaliana.