Ascorbic acid-mediated reactive oxygen species homeostasis modulates the switch from tapetal cell division to cell differentiation in Arabidopsis.

The major antioxidant L-ascorbic acid (AsA) plays important roles in plant growth, development, and stress responses. However, the importance of AsA concentration and the regulation of AsA metabolism in plant reproduction remain unclear. In Arabidopsis (Arabidopsis thaliana) anthers, the tapetum monolayer undergoes cell differentiation to support pollen development. Here, we report that a transcription factor, DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1), inhibits tapetal cell division leading to cell differentiation. We identified SKEWED5-SIMILAR 18 (SKS18) as a downstream target of TDF1. Enzymatic assays showed that SKS18, annotated as a multicopper oxidase-like protein, has ascorbate oxidase activity, leading to AsA oxidation. We also show that VITAMIN C DEFECTIVE1 (VTC1), an AsA biosynthetic enzyme, is negatively controlled by TDF1 to maintain proper AsA contents. Consistently, either knockout of SKS18 or VTC1 overexpression raised AsA concentrations, resulting in extra tapetal cells, while SKS18 overexpression in tdf1 or the vtc1-3 tdf1 double mutant mitigated their defective tapetum. We observed that high AsA concentrations caused lower accumulation of reactive oxygen species (ROS) in tapetal cells. Overexpression of ROS scavenging genes in tapetum restored excess cell divisions. Thus, our findings demonstrate that TDF1-regulated AsA balances cell division and cell differentiation in the tapetum through governing ROS homeostasis.

The temporal regulation of TEK contributes to pollen wall exine patterning.

Pollen wall consists of several complex layers which form elaborate species-specific patterns. In Arabidopsis, the transcription factor ABORTED MICROSPORE (AMS) is a master regulator of exine formation, and another transcription factor, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), specifies formation of the nexine layer. However, knowledge regarding the temporal regulatory roles of TEK in pollen wall development is limited. Here, TEK-GFP driven by the AMS promoter was prematurely expressed in the tapetal nuclei, leading to complete male sterility in the pAMS:TEK-GFP (pat) transgenic lines with the wild-type background. Cytological observations in the pat anthers showed impaired callose synthesis and aberrant exine patterning. CALLOSE SYNTHASE5 (CalS5) is required for callose synthesis, and expression of CalS5 in pat plants was significantly reduced. We demonstrated that TEK negatively regulates CalS5 expression after the tetrad stage in wild-type anthers and further discovered that premature TEK-GFP in pat directly represses CalS5 expression through histone modification. Our findings show that TEK flexibly mediates its different functions via different temporal regulation, revealing that the temporal regulation of TEK is essential for exine patterning. Moreover, the result that the repression of CalS5 by TEK after the tetrad stage coincides with the timing of callose wall dissolution suggests that tapetum utilizes temporal regulation of genes to stop callose wall synthesis, which, together with the activation of callase activity, achieves microspore release and pollen wall patterning.

Slowing development restores the fertility of thermo-sensitive male-sterile plant lines.

Temperature-sensitive genic male sterility (TGMS) lines are widely used in the breeding of hybrid crops1,2, but by what means temperature as a general environmental factor reverses the fertility of different TGMS lines remains unknown. Here, we identified an Arabidopsis TGMS line named reversible male sterile (rvms) that is fertile at low temperature (17 °C) and encodes a GDSL lipase. Cytological observations and statistical analysis showed that low temperature slows pollen development. Further screening of restorers of rvms, as well as crossing with a slow-growth line at normal temperature (24 °C), demonstrate that slowing of development overcomes the defects of rvms microspores and allows them to develop into functional pollen. Several other Arabidopsis TGMS lines were identified, and their fertility was also restored by slowing of development. Given that male reproductive development is conserved3, we propose that slowing of development is a general mechanism applicable to the sterility-fertility conversion of TGMS lines from different plant species.