Extraction of Soluble Proteins from Leaves.

Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.

Faster than expected Rubisco deactivation in shade reduces cowpea photosynthetic potential in variable light conditions.

Cowpea is the major source of vegetable protein for rural populations in sub-Saharan Africa and average yields are not keeping pace with population growth. Each day, crop leaves experience many shade events and the speed of photosynthetic adjustment to this dynamic environment strongly affects daily carbon gain. Rubisco activity is particularly important because it depends on the speed and extent of deactivation in shade and recovers slowly on return to sun. Here, direct biochemical measurements showed a much faster rate of Rubisco deactivation in cowpea than prior estimates inferred from dynamics of leaf gas exchange in other species1-3. Shade-induced deactivation was driven by decarbamylation, and half-times for both deactivation in shade and activation in saturating light were shorter than estimates from gas exchange (≤53% and 79%, respectively). Incorporating these half-times into a model of diurnal canopy photosynthesis predicted a 21% diurnal loss of productivity and suggests slowing Rubisco deactivation during shade is an unexploited opportunity for improving crop productivity.

Identification of a functionally significant tri-allelic genotype in the Tyrosinase gene (TYR) causing hypomorphic oculocutaneous albinism (OCA1B).

Oculocutaneous albinism (OCA) and ocular albinism (OA) are inherited disorders of melanin biosynthesis, resulting in loss of pigment and severe visual deficits. OCA encompasses a range of subtypes with overlapping, often hypomorphic phenotypes. OCA1 is the most common cause of albinism in European populations and is inherited through autosomal recessive mutations in the Tyrosinase (TYR) gene. However, there is a high level of reported missing heritability, where only a single heterozygous mutation is found in TYR. This is also the case for other OCA subtypes including OCA2 caused by mutations in the OCA2 gene. Here we have interrogated the genetic cause of albinism in a well phenotyped, hypomorphic albinism population by sequencing a broad gene panel and performing segregation studies on phenotyped family members. Of eighteen probands we can confidently diagnose three with OA and OCA2, and one with a PAX6 mutation. Of six probands with only a single heterozygous mutation in TYR, all were found to have the two common variants S192Y and R402Q. Our results suggest that a combination of R402Q and S192Y with a deleterious mutation in a 'tri-allelic genotype' can account for missing heritability in some hypomorphic OCA1 albinism phenotypes.

HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 is required for circadian periodicity through the promotion of nucleo-cytoplasmic mRNA export in Arabidopsis.

Cold acclimation has been shown to be attenuated by the degradation of the INDUCER OF CBF EXPRESSION1 protein by the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1). However, recent work has suggested that HOS1 may have a wider range of roles in plants than previously appreciated. Here, we show that hos1 mutants are affected in circadian clock function, exhibiting a long-period phenotype in a wide range of temperature and light environments. We demonstrate that hos1 mutants accumulate polyadenylated mRNA in the nucleus and that the circadian defect in hos1 is shared by multiple mutants with aberrant mRNA export, but not in a mutant attenuated in nucleo-cytoplasmic transport of microRNAs. As revealed by RNA sequencing, hos1 exhibits gross changes to the transcriptome with genes in multiple functional categories being affected. In addition, we show that hos1 and other previously described mutants with altered mRNA export affect cold signaling in a similar manner. Our data support a model in which altered mRNA export is important for the manifestation of hos1 circadian clock defects and suggest that HOS1 may indirectly affect cold signaling through disruption of the circadian clock.