Starch Granule Size and Morphology of Arabidopsis thaliana Starch-Related Mutants Analyzed during Diurnal Rhythm and Development

Transitory starch plays a central role in the life cycle of plants. Many aspects of this important metabolism remain unknown; however, starch granules provide insight into this persistent metabolic process. Therefore, monitoring alterations in starch granules with high temporal resolution provides one significant avenue to improve understanding. Here, a previously established method that combines LCSM and safranin-O staining for in vivo imaging of transitory starch granules in leaves of Arabidopsis thaliana was employed to demonstrate, for the first time, the alterations in starch granule size and morphology that occur both throughout the day and during leaf aging. Several starch-related mutants were included, which revealed differences among the generated granules. In ptst2 and sex1-8, the starch granules in old leaves were much larger than those in young leaves; however, the typical flattened discoid morphology was maintained. In ss4 and dpe2/phs1/ss4, the morphology of starch granules in young leaves was altered, with a more rounded shape observed. With leaf development, the starch granules became spherical exclusively in dpe2/phs1/ss4. Thus, the presented data provide new insights to contribute to the understanding of starch granule morphogenesis.

Starch Granules in Arabidopsis thaliana Mesophyll and Guard Cells Show Similar Morphology but Differences in Size and Number

Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.

Diurnal variation of transitory starch metabolism is regulated by plastid proteins WXR1/WXR3 in Arabidopsis young seedlings

Transitory starch is the portion of starch that is synthesized during the day in the chloroplast and usually used for plant growth overnight. Here, we report altered metabolism of transitory starch in the wxr1/wxr3 (weak auxin response 1/3) mutants of Arabidopsis. WXR1/WXR3 were previously reported to regulate root growth of young seedlings and affect the auxin response mediated by auxin polar transport in Arabidopsis. In this study the wxr1/wxr3 mutants accumulated transitory starch in cotyledon, young leaf, and hypocotyl at the end of night. WXR1/WXR3 expression showed diurnal variation. Grafting experiments indicated that the WXRs in root were necessary for proper starch metabolism and plant growth. We also found that photosynthesis was inhibited and the transcription level of DIN1/DIN6 (Dark-Inducible 1/6) was reduced in wxr1/wxr3. The mutants also showed a defect in the ionic equilibrium of Na+ and K+, consistent with our bioinformatics data that genes related to ionic equilibrium were misregulated in wxr1. Loss of function of WXR1 also resulted in abnormal trafficking of membrane lipids and proteins. This study reveals that the plastid proteins WXR1/WXR3 play important roles in promoting transitory starch degradation for plant growth over night, possibly through regulating ionic equilibrium in the root.

The pathway of starch synthesis in Arabidopsis thaliana leaves

Many plants accumulate transitory starch reserves in their leaves during the day to buffer their carbohydrate supply against fluctuating light conditions, and to provide carbon and energy for survival at night. It is universally accepted that transitory starch is synthesized from ADP-glucose (ADPG) in the chloroplasts. However, the consensus that ADPG is made in the chloroplasts by ADPG pyrophosphorylase has been challenged by a controversial proposal that ADPG is made primarily in the cytosol, probably by sucrose synthase (SUS), and then imported into the chloroplasts. To resolve this long-standing controversy, we critically re-examined the experimental evidence that appears to conflict with the consensus pathway. We show that when precautions are taken to avoid artefactual changes during leaf sampling, Arabidopsis thaliana mutants that lack SUS activity in mesophyll cells (quadruple sus1234) or have no SUS activity (sextuple sus123456) have wild-type levels of ADPG and starch, while ADPG is 20 times lower in the pgm and adg1 mutants that are blocked in the classical pathway of starch synthesis. We conclude that the ADPG needed for starch synthesis in leaves is synthesized primarily by ADPG pyrophosphorylase in the chloroplasts.Significance statementMutant analysis shows that sucrose synthase makes no significant contribution to transitory starch synthesis in Arabidopsis leaves, resolving a 20-year old controversy about one of the most important pathways of photosynthetic metabolism.

BIOSYNTHESIS AND DEGRADATION OF STARCH IN HIGHER PLANTS

Numerous reviews on starch biosynthesis and degradation have appeared inthe 1980s (4, 23, 39, 40, 51, 73, 100, 101, 124, 125). Here we updateestablished concepts and emphasize three topics that we consider to now meritreexamination: the significance of enzyme multiplicity, a comparison ofdegradation of reserve and transitory starch, and the localization of starchdegrading enzymes in starch-free cellular compartments of leaf tissues. Westress the cell physiological aspects of starch metabolizing enzymes.