In the absence of photo assimilation, the starch stored in the source is degraded to replenish cellular sugars in order to avoid carbon starvation. Therefore, carbon assimilation and utilization is carefully balanced for optimal plant development. Adverse environmental conditions can disrupt the normal starch and sugars levels with repercussions for the ability of the plant to sustain growth. Drought is associated with reduced starch or sugar levels in source tissues. Salinity stress can induce higher starch accumulation in the source or sink of some species, but trigger starch reduction in others. Similarly, chilling stress is associated with accelerated source-starch accumulation or degradation. These observed increases in starch or sugars may be adaptive responses for stress-survival, or may be ‘injury’ responses resulting from the under-utilization of carbon because of growth cessation, regardless, documenting these changes is necessary for a deeper understanding of plant stress response. Feeding plants with 14CO2 is useful for tracking carbon movement, and can inform on changes in carbon allocation due to stress. Available data suggests that stress generally accelerates allocation to the sinks as an adaptive response. Salinity increased flux from source to developing fruits in tomato and to the roots in transgenic rice seedlings. Water-stress elicited a similar distribution pattern in Arabidopsis, with higher 14C allocated to the roots, in beans, where 14C flux to the pods increased, and in rice, where it stimulated 14C mobilization from the stem and allocation to the grain. Additional 14C-allocation studies under varied stress conditions could help to clarify whether or not higher source-sink flux is a universal stress response.
The observed changes in local and distant carbon fluxes in plant tissues during stress result from multiple activities – epigenetic, transcriptional, post-transcriptional and post translational changes,dutch buckets occurring across different spatial and temporal scales, which must be integrated to deliver a cohesive response to stress. Te trehalose-6-phosphate/Sucrose non-Fermented Related Kinase 1 signaling cascade may function in this way. It is critical for plant survival under low carbon and energy conditions, in part through changes in starch metabolism. Te T6P/SnRK1 can also modulate source-sink interactions; therefore, key elements of this regulatory network could potentially be activated for a ‘rewiring’ of whole plant carbohydrate use under stress. Because of the many issues with respect to plant carbon use under stress that remain unresolved, our aim in this work was to investigate changes in carbon partitioning and allocation in response to short-term drought, salinity, and cold stresses. 14CO2-labeling of a single source leaf was used to map whole-plant and intra-tissue changes in carbon use, as it can provide partitioning and allocation data in the same system. Single-leaf labeling permits more accurate tracking of 14C-movement than can be obtained by exposing the entire rosette to the label.By comparing plants exposed to different stresses it may be possible to identify convergent and divergent adaptive responses associated with each unfavorable condition. Starch content was also assayed in the source leaf and the roots of the stressed plants and the data were compared to 14C-starch fluxes to identify how starch metabolism may be regulated to alter sugar distribution. Finally, the transcriptional activity of key genes in the T6P/ SnRK1 pathway was assessed to identify genes associated with changes in carbohydrate levels under abiotic stress. By integrating these data, we present one of the first comprehensive pictures of how Arabidopsis changes carbon flux under short-term environmental stress. This information could be combined with that generated from the wealth of -omics data to broaden our understanding of plant stress response.Our first aim was to investigate how plant source and sink tissues use carbon over the diurnal cycle under normal conditions. One hour before the middle of the day , a single mature, but still developing source leaf was fed with 14CO2 for 5 min. Te labeled source leaf, unlabeled sink leaves, and the roots were harvested separately at MD, at the end of the day , and at the end of the night . MD, ED and EN correspond to 6h, 12h and 24h after dawn. Te percentage of 14C distributed among the source and the sinks was determined.
Within each tissue, the incorporation of 14C into the main metabolites pools: sugars, amino acids, organic acids, starch, protein, and ‘remaining insoluble compounds’ , was established. First, we calculated the percentage of 14C distributed from the source to the sinks. During the day, ~60% of the 14C was retained in the source leaf, but by EN, the percentage of total 14C was evenly distributed among all tissues . Nighttime export of 14C from the source, and its subsequent allocation into the sinks, accounted for the re-distribution. Second, we examined the 14C partitioning between the source and sinks to create a full picture of how allocation and subsequent partitioning were altered. Partitioning in the roots was more dynamic than in the sink leaves, and this difference was amplifed most at ED . In the roots, there was increased incorporation of 14C into metabolites used for growth — i.e. sugars, amino acids, and RICs — and less into those used for storage —i.e. protein and starch — compared to the source. Te pattern of 14C-partitioning in source leaf vs. roots therefore reflected the prioritization of biological processes in each tissue type. Te other change of note occurred at EN, when both sinks incorporated less 14C into organic acids but more into starch compared to the source. This may indicate that the sinks had greater sufficiency with respect to carbon with a relatively reduced need for organic acids as sources of energy compared to the source. Finally, we examined changes in 14C-partitioning over the diurnal cycle . Data at ED and EN were compared to that generated at MD to fully assess how the day-night cycle affected carbon partitioning in different tissues. Te metabolic pools in the source leaf were variable, while those in the sinks were relatively stable. Relative to MD, there was less 14C in the sugar and starch fraction, but an almost 2-fold greater flux into organic acids at EN in the source. Organic acids may serve as the primary substrate for respiration after reductions in the sugar pool. In the roots, at EN, the 14C percentage in sugars decreased, but increased in starch. This indicates that the starch in the roots was accumulated constantly during the diurnal cycle, with more accretion during the night than the day. In contrast, in the sink leaves, the carbon flow into sugars and starch were stable at EN, but there was a 4-fold increase in the 14C partitioned into the RICs, suggestive of nighttime growth processes.How stress altered Arabidopsis carbon use over the diurnal cycle at the cellular and whole plant level was examined.
Arabidopsis seedlings were exposed to salinity stress using 100 and 200mM NaCl, to osmotic stress using 150 and 300mM mannitol, and to cold stress by exposing roots to 0 °C cold at the beginning of photoperiod. Afer 5hours of stress treatment,grow bucket a single mature source leaf was fed with 14CO2 for 5min. Sampling was done as previously described. Osmotic stress. Carbon allocation was negatively affected by osmotic stress, and the inhibition grew in severity as the stress progressed . By EN, mild and severe mannitol stress increased the percentage of 14C in the source, and decreased it in the roots . This could reffect reduced carbon export due to enhanced source activities, inhibited carbon export from the source, reduced sink strength, or a combination thereof under osmotic stress. Carbon partitioning within the source was also modulated to a greater extent than in the sinks . At MD, both mild and severe osmotic stress reduced the 14C-partitioned into starch but increased 14C-partitioning into organic acids in the source, presumably for respiratory use. Six hours later, only severe osmotic stress had this effect leading to greater 14C flux into osmoprotectants — sugars, organic acids, and amino acids — at the expense of the storage compounds . Te 14C-flux into these osmoprotectants also increased in both sinks at the expense of the RICs, with the latter decreasing drastically in the roots. Salinity stress. Te most obvious change was the percentage of 14C allocated from source leaf into roots, which decreased significantly by EN under both mild and severe NaCl stress . Te 14C-use in source leaf was more responsive to salinity compared to the sinks . Severe salinity stress decreased 14C-partitioning into starch but increased partitioning into sugars, amino acids, and organic acids during the day in the source. At MD, more 14C was partitioned into sugars in the sink leaves, but 6h later at ED the 14C in sugars was stable, with reduced flux into starch and proteins. This indicates that 12h after the stress treatment, carbon was diverted from storage and preferentially partitioned into sugars for osmoprotection. In the roots, less 14C was partitioned into the RICs at ED and EN compared to the control, which suggest a shift away from investing 14C into resources normally used for root growth under salinity. This may have led to increased 14C accumulation into sugars at the end of night because they were under-metabolized. Interestingly, proteins were the only metabolite affected by both mild and severe salinity stress in both source and sink leaves, while it was unchanged in the roots.Further, unlike sink leaves, the source had increased 14C label in protein at MD . Te changes in 14C partitioning and allocation in response to different levels of salinity stress are summarized as follows: the source leaf partitioned less 14C into storage compounds but more 14C into osmoprotectants in response to severe salinity stress; sink tissues showed a differential response to salinity stress: similar to the source leaf, the sink leaves showed reduced 14C in storage compounds, however, roots tissue had reduced 14C in structural compounds; and the amount of 14C imported into roots tissue was inhibited by salinity; this might be due to reduced sink activity, inhibited phloem transport, or a combination thereof. Cold stress. Te percentage of 14C in root tissues was significantly reduced by cold stress at the end of night, showing similarity to tissues under osmotic and salinity stress . Carbon allocation was not affected by low temperature during the day , but carbon partitioning was highly regulated in the source leaf , especially at the end of day. Te most notable difference was that the 14C-flux into starch and RICs decreased relative to the control plants. Te decrease in starch was high at MD but lessened during the diurnal cycle, while the opposite was true for the RICs, where inhibition intensified over the day. In the source, there were also higher fluxes into sugars, amino acids, and organic acids from MD to ED. Cold also triggered increased 14C into the protein pool at MD, and decreased it at ED. At EN, the 14C in RICs strongly decreased, with a corresponding strong increase in sugars. Cold stress therefore stimulated more 14C partitioning into sugars over the diurnal cycle in the source leaf. Te sinks were less affected by cold than the source. In sink leaves, there was increased carbon flow into sugars during the day and decreased carbon into starch at night, with no difference in RICs. In contrast, the roots had increased 14C in the sugar pool at night, and reduced partitioning into the RICs . This change of 14C partitioning suggests reprioritization of reserves with a greater flux towards sugars for osmoprotection at the expense of other pathways.Te 14CO2 labeling experiment showed that starch is the most dynamic metabolite pool that changed under all types of abiotic stresses used in this study. 14C-flux into starch was down-regulated by abiotic stress, and the regulation depended on the time of day and tissue type examined. Under control conditions, 14C-partitioning into starch was stable during the day but decreased at night in the source leaf . However, this pattern was disrupted under salinity and cold stress due to reduced carbon flow into starch. In contrast to the source leaf, 14C in starch in sink leaves did not change during the day even under stress. In roots, the percentage of 14C into starch normally increased by EN, and interestingly, this partitioning was maintained under osmotic stress, but not under salinity and cold stress.