Seed-produced anti-globulin VHH-Fc antibodies retrieve globulin precursors in the insoluble fraction and modulate the Arabidopsis thaliana seed subcellular morphology

Key message Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins. Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination

Chloroplasts are central players in sugar-induced leaf growth

Leaves are the plant's powerhouses, providing energy for all organs through sugar production during photosynthesis. However, sugars serve not only as a metabolic energy source for sink tissues but also as signaling molecules, affecting gene expression through conserved signaling pathways to regulate plant growth and development. Here, we describe an in vitro experimental assay, allowing one to alter the sucrose (Suc) availability during early Arabidopsis (Arabidopsis thaliana) leaf development, with the aim to identify the affected cellular and molecular processes. The transfer of seedlings to Suc-containing medium showed a profound effect on leaf growth by stimulating cell proliferation and postponing the transition to cell expansion. Furthermore, rapidly after transfer to Suc, mesophyll cells contained fewer and smaller plastids, which are irregular in shape and contain fewer starch granules compared with control mesophyll cells. Short-term transcriptional responses after transfer to Suc revealed the repression of well-known sugar-responsive genes and multiple genes encoded by the plastid, on the one hand, and up-regulation of a GLUCOSE-6-PHOSPHATE TRANSPORTER (GPT2), on the other hand. Mutant gpt2 seedlings showed no stimulation of cell proliferation and no repression of chloroplast-encoded transcripts when transferred to Suc, suggesting that GPT2 plays a critical role in the Suc-mediated effects on early leaf growth. Our findings, therefore, suggest that induction of GPT2 expression by Suc increases the import of glucose-6-phosphate into the plastids that would repress chloroplast-encoded transcripts, restricting chloroplast differentiation. Retrograde signaling from the plastids would then delay the transition to cell expansion and stimulate cell proliferation

Leaf responses to mild drought stress in natural variants of Arabidopsis

Although the response of plants exposed to severe drought stress has been studied extensively, little is known about how plants adapt their growth under mild drought stress conditions. Here, we analyzed the leaf and rosette growth response of six Arabidopsis (Arabidopsis thaliana) accessions originating from different geographic regions when exposed to mild drought stress. The automated phenotyping platform WIWAM was used to impose stress early during leaf development, when the third leaf emerges from the shoot apical meristem. Analysis of growth-related phenotypes showed differences in leaf development between the accessions. In all six accessions, mild drought stress reduced both leaf pavement cell area and number without affecting the stomatal index. Genome-wide transcriptome analysis (using RNA sequencing) of early developing leaf tissue identified 354 genes differentially expressed under mild drought stress in the six accessions. Our results indicate the existence of a robust response over different genetic backgrounds to mild drought stress in developing leaves. The processes involved in the overall mild drought stress response comprised abscisic acid signaling, proline metabolism, and cell wall adjustments. In addition to these known severe drought-related responses, 87 genes were found to be specific for the response of young developing leaves to mild drought stress

Proximal hyperspectral imaging detects diurnal and drought-induced changes in maize physiology

Hyperspectral imaging is a promising tool for non-destructive phenotyping of plant physiological traits, which has been transferred from remote to proximal sensing applications, and from manual laboratory setups to automated plant phenotyping platforms. Due to the higher resolution in proximal sensing, illumination variation and plant geometry result in increased non-biological variation in plant spectra that may mask subtle biological differences. Here, a better understanding of spectral measurements for proximal sensing and their application to study drought, developmental and diurnal responses was acquired in a drought case study of maize grown in a greenhouse phenotyping platform with a hyperspectral imaging setup. The use of brightness classification to reduce the illumination-induced non-biological variation is demonstrated, and allowed the detection of diurnal, developmental and early drought-induced changes in maize reflectance and physiology. Diurnal changes in transpiration rate and vapor pressure deficit were significantly correlated with red and red-edge reflectance. Drought-induced changes in effective quantum yield and water potential were accurately predicted using partial least squares regression and the newly developed Water Potential Index 2, respectively. The prediction accuracy of hyperspectral indices and partial least squares regression were similar, as long as a strong relationship between the physiological trait and reflectance was present. This demonstrates that current hyperspectral processing approaches can be used in automated plant phenotyping platforms to monitor physiological traits with a high temporal resolution

Levans in excised leaves of Dactylis glomerata: Effects of light, sugars, temperature and senescence

Dactylis glomerata (orchardgrass) accumulates a single series of levans and the high DP polymers might be correlated with an increased stress resistance. A single levan series could be induced in excised orchardgrass leaves, without any 1-kestose accumulation, strongly suggesting that fructan synthesis occurs independently of 1-SST activity. This elegant excised leaf system was used to study fructan metabolism regulation as affected by environmental conditions and exogenous sugar treatments. In contrast to the well-studied barley excised leaf system, fructan biosynthesis could not be rapidly induced in the light without exogenous sugar and only a limited fructan synthesis was observed in the dark with sugar. it can be concluded that both light and sugar are needed to achieve an optimal fructan synthesis. To induce fructan biosynthesis, sucrose could be replaced by a combination of glucose and fructose. Fructans were found to be a surplus pool of sucrose when a threshold sucrose concentration is surpassed. A metabolic switch to fructan degradation was observed when induced orchardgrass leaves were incubated in the dark at 30 degrees C. Interestingly, fructans persisted during senescence of sugar-induced orchardgrass leaves. On the longer term, these fundamental regulatory insights might help to create superior grasses for future feed and/or biomass production.status: publishe

DELLA Signaling Mediates Stress-Induced Cell Differentiation in Arabidopsis Leaves through Modulation of Anaphase-Promoting Complex/Cyclosome Activity1[W][OA]

Drought is responsible for considerable yield losses in agriculture due to its detrimental effects on growth. Drought responses have been extensively studied, but mostly on the level of complete plants or mature tissues. However, stress responses were shown to be highly tissue and developmental stage specific, and dividing tissues have developed unique mechanisms to respond to stress. Previously, we studied the effects of osmotic stress on dividing leaf cells in Arabidopsis (Arabidopsis thaliana) and found that stress causes early mitotic exit, in which cells end their mitotic division and start endoreduplication earlier. In this study, we analyzed this phenomenon in more detail. Osmotic stress induces changes in gibberellin metabolism, resulting in the stabilization of DELLAs, which are responsible for mitotic exit and earlier onset of endoreduplication. Consequently, this response is absent in mutants with altered gibberellin levels or DELLA activity. Mitotic exit and onset of endoreduplication do not correlate with an up-regulation of known cell cycle inhibitors but are the result of reduced levels of DP-E2F-LIKE1/E2Fe and UV-B-INSENSITIVE4, both inhibitors of the developmental transition from mitosis to endoreduplication by modulating anaphase-promoting complex/cyclosome activity, which are down-regulated rapidly after DELLA stabilization. This work fits into an emerging view of DELLAs as regulators of cell division by regulating the transition to endoreduplication and differentiation

A Reciprocal N-15-labeling proteomic analysis of expanding arabidopsis leaves subjected to osmotic stress indicates importance of mitochondria in preserving plastid functions

Plants respond to environmental stress by dynamically reprogramming their growth. Whereas stress onset is accompanied by rapid growth inhibition leading to smaller organs, growth will recover and adapt once the stress conditions become stable and do no threaten plant survival. Here, adaptation of growing Arabidopsis thaliana leaves to mild and prolonged osmotic stress was investigated by means of a complete metabolic labeling strategy with the N-15-stable isotope as a complement to a previously published transcript and metabolite profiling. Global analysis of protein changes revealed that plastidial ATPase, Calvin cycle, and photorespiration were down-regulated, but mitochondrial ATP synthesis was up-regulated, indicating the importance of mitochondria in preserving plastid functions during water stress. Although transcript and protein data correlated well with the stable and prolonged character of the applied stress, numerous proteins were clearly regulated at the post-transcriptional level that could, at least partly, be related to changes in protein synthesis and degradation. In conclusion, proteomics using the N-15 labeling helped understand the mechanisms underlying growth adaptation to osmotic stress and allowed the identification of candidate genes to improve plant growth under limited water