Transgenic lines of sugarcane with increased total sugar content have been obtained in previous work in our lab, through the introduction of a sucrose isomerase (SI) gene designed vacuole-targeting of the SI gene product. Some of the resulting transgenic lines accumulate the high value sugar isomaltulose (IM) without decrease in stored sucrose content. Other lines show enhanced sucrose content with low levels of IM in mature storage tissues. Under containment glasshouse conditions, these “SugarBooster™” lines have shown up to two-fold increase in the total sugar concentration in the harvested juice. This remarkable step above the former ceiling in stored sugar concentration may permit new insights into the mechanisms by which plants regulate sugar accumulation, a pivotal question in plant biology.
Studies conducted in this thesis are a part of the effort to understand the underlying mechanisms. Experiments were focused on the regulation of SI and endogenous genes related to sucrose metabolism, as revealed by transcript levels. Enzyme activity was also tested for sucrose phosphate synthase (SPS), a key enzyme in plant sucrose biosynthesis.
Initially, the relationship between IM content and SI transcript level was analyzed. Real-time quantitative polymerase chain reaction (RT-qPCR) successfully detected SI gene transcripts in internodes of different maturities in SugarBooster™ lines, but no correlation was found between the transcript level and IM content. In contrast, northern blotting to distinguish full-length SI mRNA from degraded transcripts revealed a positive relationship between IM content and functional transcripts of SI. The results imply that stabilization of SI mRNA is important for high IM content but that high sucrose accumulation accompanied by low IM involves other mechanisms.
Therefore, transcript levels of several key genes in sucrose metabolism were analyzed in internodes at various developmental stages in SugarBooster™ and control lines. These genes encode sucrose phosphate synthase (SPS), sucrose synthase (SuSy), soluble acid invertase (SAI), neutral invertase (NI), cell wall invertase (CWI), a putative sugar transporter type 2a (Type2a) and sucrose transporter type 6 (Type6). Sucrose content was negatively correlated to the transcript levels of SAI, but there were no significant correlations between sugar content and mRNA levels of other tested enzymes across all developmental stages. However, interesting transcript patterns that might contribute to high sucrose accumulation were observed in some lines. Examples from different lines include: SuSy transcripts increased in immature internodes but decreased in mature ones; NI expression decreased in mature internodes and sugar transporters increased in some cases.
Further analysis was undertaken on transcript regulation of five SPS families, as SPS is the rate-limiting enzyme for sucrose biosynthesis in some plant systems. As recently reported from a CSIRO analysis of sugarcane progeny segregating for sucrose content, transcripts of SPS family 2 predominated in the stem for both SugarBooster™ and control lines. Transcripts of SPS families 3 and 4 were less abundant and families 1 and 5 were rare. More abundant mRNAs of SPS families 2, 3 and 4 indicate that they play an important role in sucrose accumulation in the stem. However, none of the mRNA levels of the five families had a significant correlation to the SPS enzyme activity or to the sucrose content measured in corresponding tissues. In contrast, SPS enzyme activity was found positively correlated to sucrose content. It revealed that regulation of SPS in sugarcane is mainly at the level of enzyme activity.
These studies have been constrained by the availability of experimental material in early vegetative generations of SugarBooster™ lines under containment glasshouse conditions. As the opportunity emerges for more material from approved field trials, it will be important to identify the most stable lines under field conditions, for further studies on metabolites, carbon partitioning and enzyme activity to fill the gap in understanding between regulation at transcript level and the SugarBooster™ phenotypes.