Current methods of in-house plant phenotyping are providing a powerful new tool for plant biology studies. The self-constructed and commercial platforms established in the last few years, employ non-destructive methods and measurements on a large and high-throughput scale. The platforms offer to certain extent, automated measurements, using either simple single sensor analysis, or advanced integrative simultaneous analysis by multiple sensors. However, due to the complexity of the approaches used, it is not always clear what such forms of plant phenotyping can offer the potential end-user, i.e. plant biologist. This review focuses on imaging methods used in the phenotyping of plant shoots including a brief survey of the sensors used. To open up this topic to a broader audience, we provide here a simple introduction to the principles of automated non-destructive analysis, namely RGB, chlorophyll fluorescence, thermal and hyperspectral imaging. We further on present an overview on how and to which extent, the automated integrative in-house phenotyping platforms have been used recently to study the responses of plants to various changing environments.

Recently emerging approaches to high-throughput plant phenotyping have discovered their importance as tools in unravelling the complex questions of plant growth, development and response to the environment, both in basic and applied science. High-throughput methods have been also used to study plant responses to various types of biotic and abiotic stresses (drought, heat, salinity, nutrient-starving, UV light) but only rarely to cold tolerance. We present here an experimental procedure of integrative high-throughput in-house phenotyping of plant shoots employing automated simultaneous analyses of shoot biomass and photosystem II efficiency to study the cold tolerance of pea (Pisum sativum L.). For this purpose, we developed new software for automatic RGB image analysis, evaluated various parameters of chlorophyll fluorescence obtained from kinetic chlorophyll fluorescence imaging, and performed an experiment in which the growth and photosynthetic activity of two different pea cultivars were followed during cold acclimation. The data obtained from the automated RGB imaging were validated through correlation of pixel based shoot area with measurement of the shoot fresh weight. Further, data obtained from automated chlorophyll fluorescence imaging analysis were compared with chlorophyll fluorescence parameters measured by a non-imaging chlorophyll fluorometer. In both cases, high correlation was obtained, confirming the reliability of the procedure described. This study of the response of two pea cultivars to cold stress confirmed that our procedure may have important application, not only for selection of cold-sensitive/tolerant varieties of pea, but also for studies of plant cold-response strategies in general. The approach, provides a very broad tool for the morphological and physiological selection of parameters which correspond to shoot growth and the efficiency of photosystem II, and is thus applicable in studies of various plant species and crops.

Cytokinin-mediated responses of Arabidopsis to volatile compounds emitted by pathogenic microorganisms involve the activation of plastidic phosphoglucose isomer

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.