The initiation of starch granule formation is still poorly understood. However, the soluble starch synthase 4 (SS4) appears to be a major component of this process since it is required to synthesize the correct number of starch granules in the chloroplasts of Arabidopsis thaliana plants. A yeast two-hybrid screen allowed the identification of several putative SS4 interacting partners. We identified the product of At4g32190 locus as a chloroplast-targeted PROTEIN INVOLVED IN STARCH INITIATION (named PII1). Arabidopsis mutants devoid of PII1 display an alteration of the starch initiation process and accumulate, on average, one starch granule per plastid instead of the five to seven granules found in plastids of wild-type plants. These granules are larger than in wild-type, and they remain flat and lenticular. pii1 mutants display wild-type growth rates and accumulate standard starch amounts. Moreover, starch characteristics, such as amylopectin chain length distribution, remain unchanged. Our results reveal the involvement of PII1 in the starch priming process in Arabidopsis leaves through interaction with SS4.
24The initiation of starch granule formation is still poorly understood. However, soluble starch 25 synthase 4 (SS4) appears to be a major component of this process since it is required to 26 synthetize the correct number of starch granules in the chloroplasts of Arabidopsis thaliana 27 plants. A yeast-2-hybrid screen allowed the identification of several putative SS4 interacting 28 partners. We identified the product of At4g32190 locus as a chloroplast-targeted PROTEIN 29 INVOLVED IN STARCH INITIATION (named PII1). Arabidopsis mutants devoid of PII1 30display an alteration of starch initiation process and accumulate, on average, one starch 31 granule per plastid instead of the 5 to 7 granules found in plastids of wild-type plants. These 32 granules are larger than in wild type and they remain flat and lenticular. pii1 mutants display 33 wild-type growth rates and accumulate standard starch amounts. Moreover, starch 34 characteristics, such as amylopectin chain length distribution, remain unchanged. Our results 35 reveal the involvement of PII1 in starch priming process in Arabidopsis leaves through 36 interaction with SS4. 37 38 39 Keywords: starch, starch priming, starch granule size, starch initiation, SS4, PII1, Arabidopsis 40 41 42 43 phenocopy of that of the ss4 mutant, because plant growth and starch granule morphology are 110 unaltered in pii1 mutant compared to wild type. We propose that PII1 is required for starch 111 granule initiation by controlling the catalytic activity of SS4. 112 113 Material and methods 114 115 Plant material and growth conditions 116The ULTImate Y2H TM was carried out by Hybrigenics-services (Paris, France) using 117 SS4 as bait (amino acids 43 to 1040) against a library prepared from one-week-old seedlings. 118Among 125 millions interaction tested, 369, corresponding to 80 different proteins, were 119 positives. Using the ChloroP algorithm prediction (Emanuelsson et al., 1999), we were able to 120 select proteins with predicted chloroplast targeting peptides. We ended-up with six candidates 121 among which PII1 (At4g32190) was selected (Table S1). Hybrigenics-services provides 122interaction results associated to a predicted biological score (PBS). This score indicates the 123 interaction reliability and is divided in 6 different classes (A to F): A: very high confidence in 124 the interaction. B: high confidence in the interaction. C: good confidence of interaction. D: 125 moderate confidence of interaction. E: interaction involving highly connected prey domains. 126This class is subjected to non-specific interactions. F: experimentally proven artifacts. 127 Arabidopsis thaliana lines were obtained from NASC (Nottingham Arabidopsis Stock 128Centre; http://Arabidopsis.info; (Alonso et al., 2003)) or from the collection generated at 129 URGV (INRA of Versailles; (Samson et al., 2002)). Wassilewskija (Ws) and Columbia (Col-130 0) lines were used as wild type references. T-DNA insertion lines used are: pii1-1 131 (SALK_122445); pii1-2 (FLAG_137A02); ss4-1 (GABI_290D11); ss4-2 (FLA...
Starch granules that accumulate in the plastids of plants vary in size, shape, phosphate, or protein content according to their botanical origin. Depending on their size, the applications in food and nonfood industries differ. Being able to master starch granule size for a specific plant, without alteration of other characteristics (phosphate content, protein content, etc.), is challenging. The development of a simple and effective screening method to determine the size and shape of starch granules in a plant population is therefore of prime interest. In this study, we propose a new method, NegFluo, that combines negative confocal autofluorescence imaging in leaf and machine learning (ML)-based image analysis. It provides a fast, automated, and easy-to-use pipeline for both in situ starch granule imaging and its morphological analysis. NegFluo was applied to Arabidopsis leaves of wild-type and ss4 mutant plants. We validated its accuracy by comparing morphological quantifications using NegFluo and state-of-the-art methods relying either on starch granule purification or on preparation-intensive electron microscopy combined with manual image analysis. NegFluo thus opens the way to fast in situ analysis of starch granules.
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