Genetic and isotope ratio mass spectrometric evidence for the occurrence of starch degradation and cycling in illuminated Arabidopsis leaves

Abstract

Although there is a great wealth of data supporting the occurrence of simultaneous synthesis and breakdown of storage carbohydrate in many organisms, previous 13CO2 pulse-chase based studies indicated that starch degradation does not operate in illuminated Arabidopsis leaves. Here we show that leaves of gwd, sex4, bam4, bam1/bam3 and amy3/isa3/lda starch breakdown mutants accumulate higher levels of starch than wild type (WT) leaves when cultured under continuous light (CL) conditions. We also show that leaves of CL grown dpe1 plants impaired in the plastidic disproportionating enzyme accumulate higher levels of maltotriose than WT leaves, the overall data providing evidence for the occurrence of extensive starch degradation in illuminated leaves. Moreover, we show that leaves of CL grown mex1/pglct plants impaired in the chloroplastic maltose and glucose transporters display a severe dwarf phenotype and accumulate high levels of maltose, strongly indicating that the MEX1 and pGlcT transporters are involved in the export of starch breakdown products to the cytosol to support growth during illumination. To investigate whether starch breakdown products can be recycled back to starch during illumination through a mechanism involving ADP-glucose pyrophosphorylase (AGP) we conducted kinetic analyses of the stable isotope carbon composition (δ13C) in starch of leaves of 13CO2 pulsed-chased WT and AGP lacking aps1 plants. Notably, the rate of increase of δ13C in starch of aps1 leaves during the pulse was exceedingly higher than that of WT leaves. Furthermore, δ13C decline in starch of aps1 leaves during the chase was much faster than that of WT leaves, which provides strong evidence for the occurrence of AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis leaves.

Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day

“…Our conclusions from 13 CO 2 labeling experiments are at odds with those of Baslam et al (2017), who measured 13 C enrichment of starch (d 13 C) following pulse-chase experiments with 13 CO 2 from ZT4 onwards. These authors concluded that substantial starch degradation and resynthesis occurs at the same time as new synthesis throughout light periods.…”

“…These authors concluded that substantial starch degradation and resynthesis occurs at the same time as new synthesis throughout light periods. We discuss why our conclusions are different from those of Baslam et al (2017) in Supplemental Text S1.…”

“…Simultaneous synthesis and degradation of starch in the light was observed in spinach (Spinacia oleracea) chloroplasts, following a protocol for chloroplast preparation that involved illumination for 14 to 16 h (Stitt and Heldt, 1981). Arabidopsis mutants with deficiencies in starch degradation accumulate higher levels of starch in constant light than wild-type plants, implying that starch levels in these conditions are determined by simultaneous synthesis and degradation (Caspar et al, 1991;Baslam et al, 2017). In triose phosphate transporter mutants of Arabidopsis and tobacco, the block in export of carbon from the chloroplast in the light is bypassed through starch turnover and export of starch degradation products (Häusler et al, 1998;Walters et al, 2004).…”

Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium conductrix SAG 241.80: implications to maltose excretion by a green alga

“…Fundamental to this dogma is the idea that processes associated with starch degradation are essentially turned off during the light period. This belief has been challenged to some degree by multiple reports that provide evidence that starch is being actively synthesized and degraded in plant cells during illumination (Stitt and Heldt, ; Lozovaya et al ., ; Muñoz et al ., ; Baslam et al ., ). The question of whether starch can be synthesized and degraded simultaneously is important, because maltose production through the well‐characterized pathway of starch degradation provides the simplest and most direct pathway to maltose within the cytosol (Lu and Sharkey, ; Streb and Zeeman, ).…”

NTRC and Thioredoxin f Overexpression Differentially Induces Starch Accumulation in Tobacco Leaves

“…ADP-glucose pyrophosphorylase (AGPase), which catalyzes the first committed step of starch synthesis in higher plants, is considered to be a key enzyme in this process, controlling the flux of carbon into starch [18]. Additionally, an alternative model that considers the transport of ADP-glucose (generated in the cytosol through sucrose synthase activity) into the chloroplast has also been proposed [19][20][21]. Three major classes of enzymes act simultaneously in starch synthesis: starch synthases (SSs), branching enzymes (BEs), and debranching enzymes (DBEs).…”