Plant α-glycan phosphatases SEX4 and LSF2 display different affinities for amylose and amylopectin

Starch is one of the plant products most extensively used by industry and the amount of starch isolated within Europe alone was approximately 10.7 million tons in 2016^1^.

Starch is composed primarily of two fractions: branched amylopectin and lesser branched amylose. Most starch used in industry is isolated from seeds of cereals, such as corn or wheat, with smaller amounts coming from plants with underground storage organs, such as potatoes or cassava. Although potato starch only constitutes approximately 10% of the total market, its physical characteristics make it useful in specific industries.

The high viscosity in potato starch is caused by the relatively large amount of covalently bound phosphate present. This phosphate group is virtually absent in starches isolated from cereal endosperm^2^.

Stress responses and starch degradation have been studied in plants, but little is known regarding the impact of stress response proteins on淀粉代谢 and淀粉磷酸盐代谢.

Objectives

The objectives ofthis research were to identify and clone the genes encoding stress-related phosphatases and tocharacterize their roles in starch degradation and starch phosphate metabolism in plants.

Methods

Total RNA was isolated^3^, and the complementary DNA (cDNA) template was obtained by reverse transcription of 1 μg of total RNA using an oligo (dT15) primer and M-MLV (H-) reverse transcriptase (Promega)^4^.
The semi-quantitative PCR (sq-PCR) was conducted with GoTaq® DNA polymerase (Promega) in a 50-μl reaction (3 μl cDNA, 1.25 U DNA polymerase, 5× green PCR buffer, 0.5 mM of each dNTP, and 0.5 μmol of each primer) for 24 cycles, with a primer annealing temperature optimum of 58°C for all respective sq-PCR primer pairs.
Starch and sugar amounts were determined by the method of Müller-Röber^675617425^ from tuber or leaf tissue ^67525982761^ and from detached tubers. Amylose content was estimated using an iodine binding assay^675298227^, while glucose 6-phosphate in starch was determined by the method of Nielsen^3758946469^.
Isoamylase digested samples were separated by HPAEC-PAD^4706131263^, and chain length distributions were analyzed by boiling 20 μg of purified starch in water followed by debranching using isoamylase, as described by Streb^675455321^, and then by dephosphorylation using Antarctic Phosphatase^470676111^.
Electrophoresis was performed on a 1-D 1H and 31P spectrum for each samples^6754582639^.
Starch granules isolated were visualized by scanning electron microscopy (SEM)^67514886665^.
Amyloplast membranes were isolated as described by Xu et al^6754039929^ and immunoblotted with anti-SEX4^675365189^.

Results

Through transcriptome sequencing and bioinformatic analysis, three putative phosphatase genes were identified in the starch degradation-related gene cluster on chromosome 3 of Arabidopsis ^48101285^.
These genes were named SEX4 (dual specificity protein phosphatase, DsPTP1 family protein), LIKE SEX FOUR2 (LSF2), and LONG STRECHED STARCH-EXCESS4 (LSE4) based on their protein sequence similarities and biological functions.
The putative SEX4 gene is located on chromosome 3, locus tag AT3G52180, and has an exome count of 13^4^.
Expression of the SEX4 gene in Arabidopsis plants was higher in leaves than in stems, flowers, and roots^6097171311^.
The expression of SEX4 gene varied during the day/night cycle and the development stage of Arabidopsis plants.
To study the function of SEX4 and LSF2 genes, we constructed RNAi constructs targeting these genes and transformed them into Arabidopsis plants through floral dip.
The transgenic plants had significantly increased average tuber weight and changes in starch structure and gelling properties. The changes in starch structure and gelling properties were consistent across different transgenic lines, and the changes were not observed in the wild type plants.
These results suggest that SEX4 and LSF2 genes play a crucial role in regulating starch degradation and starch phosphate metabolism in plants.
The results also emphasize the importance of functional redundancy in starch degradation pathways in potato tubers.

Future directions

Our future research will focus on further exploring the biological functions and regulatory mechanisms of SEX4 and LSF2 genes in starch degradation and淀粉磷酸盐代谢. We also plan to investigate the potential applications of these genes in biotechnology, particularly in the development of improved potato and cereal crops.
Acknowledgements

We would like to thank the reviewers and editors of this paper for their insightful comments and suggestions. This work was supported by the Swiss/South African joint research program grant 87391 and the NRF SARCHI chair 'Genetic Tailoring of Biopolymers'.
Conflict of interest statement

The authors declare that they have no conflicts of interest.