Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element–specific proteins reveal differentiation of the endomembrane system

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins (SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Cell Wall

The application of AuNPs on the infected barley cultivar had great damage results on Barley Yellow Dwarf Virus (BYDV-PAV) particles in TEM. Observation of TEM images provided an insight into the transport of AuNPs through the plasmodesmata endoplasmic reticulum route, where they likely accumulated as the channels narrowed. The cytoplasmic parenchyma cell components do not have an intact peripheral location, but taking irregular shapes, internal movement between adjacent two cells seems to be the VLPs moved toward via plasmodesmata. TEM micrographs; showing different abnormalities in the cell wall due to viral infection. Application of AuNPs revealed sticky Integrated AuNPs inside the cell wall with low and high density. The mechanical transportation of the virus through the sieve elements with endosomes was observed. The mechanical transportation of virus particles through the cell wall with some vesicles, amorphous inclusions, and filamentous particles was proved through the sieve elements with filamentous strands.

Metaphloem development in the Arabidopsis root tip

The phloem transport network is a major evolutionary innovation that enabled plants to dominate terrestrial ecosystems. In the growth apices, the meristems, apical stem cells continuously produce early, so-called protophloem. This is easily observed in Arabidopsis root meristems, where the differentiation of individual protophloem sieve element precursors into interconnected, conducting sieve tubes is laid out in a spatio-temporal gradient. The mature protophloem eventually collapses as the neighboring metaphloem takes over its function further distal from the stem cell niche. Compared to protophloem, metaphloem ontogenesis is poorly characterized, primarily because its visualization is challenging. Here we describe the improved TetSee protocol to investigate metaphloem development in Arabidopsis root tips in combination with a set of molecular markers. We found that mature metaphloem sieve elements are only observed in the late post-meristematic root although their specification is initiated as soon as protophloem sieve elements enucleate. Moreover, unlike protophloem sieve elements, metaphloem sieve elements only differentiate once they have fully elongated. Finally, our results suggest that metaphloem differentiation is not directly controlled by protophloem-derived cues but rather follows a distinct, robust developmental trajectory.

Metaphloem development in the Arabidopsis root tip

The phloem transport network is a major evolutionary innovation that enabled plants to dominate terrestrial ecosystems. In the growth apices, the meristems, apical stem cells continuously produce early, so-called protophloem. This is easily observed in Arabidopsis root meristems, where the differentiation of individual protophloem sieve element precursors into interconnected, conducting sieve tubes is laid out in a spatio-temporal gradient. The mature protophloem eventually collapses as the neighboring metaphloem takes over its function further distal from the stem cell niche. Compared to protophloem, metaphloem ontogenesis is poorly characterized, primarily because its visualization is challenging. Here we describe an improved protocol to investigate metaphloem development in Arabidopsis root tips in combination with a set of new molecular markers. We found that mature metaphloem sieve elements are only observed in the late post-meristematic root although their specification is initiated as soon as protophloem sieve elements enucleate. Moreover, unlike protophloem sieve elements, metaphloem sieve elements only differentiate once they have fully elongated. Finally, our results suggest that metaphloem differentiation is not directly controlled by protophloem-derived cues but rather follows a distinct, robust developmental trajectory.

Role of the SUT1 and SUT2 sugar transporters during stolbur phytoplasma infection in tomato

SummaryPhytoplasmas inhabit phloem sieve elements and cause abnormal growth and altered sugar partitioning. But how they interact with phloem functions is not clearly known. The phloem responses were investigated in tomato infected by ‘Candidatus Phytoplasma solani’, at the beginning of the symptomatic stage of infection, both in symptomatic and asymptomatic leaves, the first symptoms appearing in the sink top leaf at the stem apex. Antisense lines impaired in the phloem sucrose transporters SUT1 and SUT2 were included. The infection in source leaves was not associated with symptoms. In the symptomatic, sink leaf, yellowing and leaf curling was associated with higher starch accumulation and expression of defense genes. The transcriptional analysis of symptomatic leaf midribs indicated that transcript levels for genes acting in the glycolysis and peroxisome metabolism in infected plants differed from these in non-infected plants. Phytoplasma multiplied actively in at least three additional lower leaves although they were symptomless, with no sign of activation of defense markers, although the rate of exudation of sucrose from these symptomless, source leaves was lower for infected plants. A few metabolites in phloem-enriched exudate were affected by the infection, such as glycolate and aspartate, and some of them were also affected in the control SUT1- and SUT2- antisense lines, in which sucrose retrieval or release in the sieve elements are impaired. A metabolic switch could explain the delivery of more glycolate into the sieve elements of infected plants. The findings suggest a link between sugar transport and redox homeostasis.One sentence summaryAn impairment of sucrose retrieval and release in the sieve elements occurs during phytoplasma infection, associated with changes in sugar and peroxisome metabolism

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.