Evidence for a specific and critical role of mitogen‐activated protein kinase 20 in uni‐to‐binucleate transition of microgametogenesis in tomato

Abstract

Mitogen-activated protein kinases (MAPKs) regulate diverse aspects of plant growth. However, their potential role in reproductive development remains elusive. Here, we discovered an unique role of SlMPK20, a plant-specific group D MAPK, in pollen development in tomato. RNAi-mediated suppression of SlMPK20 or its knockout using CRISPR/Cas9 significantly reduced or completely abolished pollen viability, respectively, with no effects on maternal fertility. Cell biology and gene expression analyses established that SlMPK20 exerts its role specifically at the uni-to-binucleate transition during microgametogenesis. This assertion is based on the findings that the transgenic pollen was largely arrested at the binucleate stage with the appearance of subcellular abnormality at the middle uninucleate microspore stage; and SlMPK20 mRNA and SlMPK20-GUS signals were localized in the tetrads, uninuclear microspores and binuclear pollen grains but not in microspore mother cells or mature pollen grains. Transcriptomic and proteomic analyses revealed that knockout of SlMPK20 significantly reduced the expression of a large number of genes controlling sugar and auxin metabolism and signaling in anthers. Finally, protein-protein interaction assays identified SlMYB32 as a putative target protein of SlMPK20. We conclude that SlMPK20 specifically regulates post-meiotic pollen development through modulating sugar and auxin metabolism and signaling.

The GAMYB-like gene SlMYB33 mediates flowering and pollen development in tomato

“…Lin7 has been reported to be predominately expressed in tomato anthers 63 ; however, its biological function is still elusive. Chen et al 35 speculated that Lin7 potentially plays a role in postmeiotic pollen development, while our data revealed a possible effect of Lin7 on pollen maturity in tomato, suggesting that the function of CWIN homologs in pollen development may be conserved. Moreover, one SPS and two SUS genes were obviously downregulated in transgenic anthers (Table 1).…”

“…Carbohydrate metabolism was then classified into 13 subgroups (Fig. 8b), among which starch and sucrose metabolism are essential for pollen development, especially for pollen maturity [35][36][37][38][39] . Further examination revealed a dramatic reduction in the transcript levels of most of the genes grouped into the starch and sucrose metabolism pathways (Table 1).…”

“…The two amplified fragments were cloned into the pFGC1008 vector in the reverse orientation. Then, the resulting SlMYB33-RNAi vector was transformed into tomato as described by Chen et al 35 . The positive plants were verified through PCR testing.…”

“…Samples of flower buds containing various developmental stages of pollen 24,35 were fixed, embedded, transversely sectioned (5-μm thick), dewaxed, and stained as previously described 75 . The sections were stained with 1% sarranine and 0.5% Fast Green solution and viewed under a light microscope (Olympus BX51, Tokyo, Japan) equipped with a digital camera (Olympus DP72, Tokyo, Japan).…”

Trait discovery and editing in tomato

“…In the recent years, many tomato genes have been successfully targeted by CRISPR/Cas9, resulting in alteration of traits such as root development (Ron et al ., ), leaf development (Brooks et al ., ; Pan et al ., ), day neutrality and yield (Soyk et al ., ), meristem size and inflorescence architecture (Xu et al ., ), pollen development (Chen et al ., ; Qin et al ., ), fruit parthenocarpy (Klap et al ., ), fruit ripening (Ito et al ., ; Li et al ., ), fruit composition (Nonaka et al ., ), fruit shelf life (Yu et al ., ), chilling temperature tolerance (Li et al ., ) as well as in enhanced resistance to the fungal pathogen powdery mildew (Nekrasov et al ., ). In addition, the combination of natural and gene‐edited mutations of regulators of meristem maturation successfully enabled the development of weakly branched tomato hybrids with higher flower and fruit production (Soyk et al ., ).…”