Protein biomarkers for root length and root dry mass on chromosomes 4A and 7A in wheat.

Improving the wheat (Triticum aestivum L.) root system is important for enhancing grain yield and climate resilience. Total root length (RL) and root dry mass (RM) significantly contribute to water and nutrient acquisition directly impacting grain yield and stress tolerance. This study used label-free quantitative proteomics to identify proteins associated with RL and RM in wheat near-isogenic lines (NILs). NIL pair 6 had 113 and NIL pair 9 had 30 differentially abundant proteins (DAPs). Three of identified DAPs located within the targeted genomic regions (GRs) of NIL pairs 6 (qDT.4A.1) and 9 (QHtscc.ksu-7A), showed consistent gene expressions at the protein and mRNA transcription (qRT-PCR) levels for asparagine synthetase (TraesCS4A02G109900), signal recognition particle 19 kDa protein (TraesCS7A02G333600) and 3,4-dihydroxy-2-butanone 4-phosphate synthase (TraesCS7A02G415600). This study discovered, for the first time, the involvement of these proteins as candidate biomarkers for increased RL and RM in wheat. However, further functional validation is required to ascertain their practical applicability in wheat root breeding. SIGNIFICANCE OF THE STUDY: Climate change has impacted global demand for wheat (Triticum aestivum L.). Root traits such as total root length (RL) and root dry mass (RM) are crucial for water and nutrient uptake and tolerance to abiotic stresses such as drought, salinity, and nutrient imbalance in wheat. Improving RL and RM could significantly enhance wheat grain yield and climate resilience. However, breeding for these traits has been limited by lack of appropriate root phenotyping methods, advanced genotypes, and the complex nature of the wheat genome. In this study, we used a semi-hydroponic root phenotyping system to collect accurate root data, near-isogenic lines (NILs; isolines with similar genetic backgrounds but contrasting target genomic regions (GRs)) and label-free quantitative proteomics to explore the molecular mechanisms underlying high RL and RM in wheat. We identified differentially abundant proteins (DAPs) and their molecular pathways in NIL pairs 6 (GR: qDT.4A.1) and 9 (GR: QHtscc.ksu-7A), providing a foundation for further molecular investigations. Furthermore, we identified three DAPs within the target GRs of the NIL pairs with differential expression at the transcript level, as confirmed by qRT-PCR analysis which could serve as candidate protein biomarkers for RL and RM improvement.

Autophagy mutants show delayed chloroplast development during de-etiolation in carbon limiting conditions.

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation conditions and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12 h post-illumination. We observed that while gene expression for photosynthetic machinery during de-etiolation was largely unaffected in atg mutants, several genes involved in photosystem assembly were transcriptionally downregulated. We also investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in chloroplast development. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected de-etiolation. Moreover, we showed that lower levels of carbon resources exacerbated the slow greening phenotype whereas higher levels of carbon resources had an opposite effect. This work suggests a lack of autophagy machinery limits chloroplast development during de-etiolation, and this is exacerbated by limited lipid turnover (lipophagy) that physically or energetically restrains chloroplast development.

Proteomics for Autophagy Receptor and Cargo Identification in Plants.

Autophagy is a catabolic process facilitating the degradation of cytoplasmic proteins and organelles in a lysosome- or vacuole-dependent manner in plants, animals, and fungi. Proteomic studies have demonstrated that autophagy controls and shapes the proteome and has identified both receptor and cargo proteins inside autophagosomes. In a smaller selection of studies, proteomics has been used for the analysis of post-translational modifications that target proteins for elimination and protein-protein interactions between receptors and cargo, providing a better understanding of the complex regulatory processes controlling autophagy. In this perspective, we highlight how proteomic studies have contributed to our understanding of autophagy in plants against the backdrop of yeast and animal studies. We then provide a framework for how the future application of proteomics in plant autophagy can uncover the mechanisms and outcomes of sculpting organelles during plant development, particularly through the identification of autophagy receptors and cargo in plants.

Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase.

Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.

Micro-respiratory measurements in plants.

Respiratory measurement in plants is one of the commonly used techniques to assess metabolic activity and in vivo redox state of plant mitochondria. However, respiration rate monitoring of Arabidopsis (Arabidopsis thaliana) remains a challenge for researchers due to the small size of its organs. In this chapter we introduce adaptations to micro-respiratory technologies to study three tissues of special interest to plant biologists: leaf sections, root tips, and seeds in this model plant species. This assay opens up new possibilities to screen and study mutants and to identify differences in ecotypes or populations of plants.