Deficiency of AtGFAT1 activity impairs growth, pollen germination and tolerance to tunicamycin in Arabidopsis.

The hexosamine biosynthetic pathway (HBP) plays essential roles in growth and development in plants. However, insight into the biological function of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), mediating the first regulatory step of the HBP, remains unclear in plants. Here, we report the molecular characterization of Arabidopsis AtGFAT1 gene. AtGFAT1 was highly expressed in mature pollen grains, but its expression was not detectable in the rest of the organs. Pollen grains bearing the gfat1-2 knockout allele displayed defects in a polar deposition of pectin and callose in the pollen cell wall, leading to no genetic transmission of the gfat1-2 allele through the male gametophyte. AtGFAT1 overexpression increased glucosamine (GlcN) content and enhanced resistance to tunicamycin (Tm) treatment, while RNAi-mediated suppression reduced GlcN content and resistance to Tm treatment. However, the decrease in Tm resistance by RNAi suppression of AtGFAT1 was recovered by a GlcN supplement. The exogenous GlcN supplement also rescued gfat1-2/gaft1-2 mutant plants, which were otherwise not viable. The gfat1-2/gfat1-2 plants stopped growing at the germination stage on GlcN-free medium, but GlcN supplement allowed wild-type growth of gfat1-2/gfat1-2 plants. In addition, reactive oxygen species production, cell death and a decrease in protein N-glycosylation were observed in gfat1-2/gaft1-2 mutant plants grown on GlcN-free medium, whereas these aberrant defects were not detectable on GlcN-sufficient medium. Taken together, these results show that the reduction of protein N-glycosylation was at least partially responsible for many aberrant phenotypes in growth and development as well as the response to Tm treatment caused by AtGFAT1 deficiency in Arabidopsis.

Systematic deletion of the ER lectin chaperone genes reveals their roles in vegetative growth and male gametophyte development in Arabidopsis.

Calnexin (CNX) and calreticulin (CRT) are homologous lectin chaperones in the endoplasmic reticulum (ER) that facilitate glycoprotein folding and retain folding intermediates to prevent their transit via the secretary pathway. The Arabidopsis genome has two CNX (CNX1 and CNX2) and three CRT (CRT1, CRT2 and CRT3) homologs. Despite growing evidence of the biological roles of CNXs and CRTs, little is understood about their function in Arabidopsis growth and development under normal conditions. Here, we report that the deletion of CNX1, but not of CNX2, in the crt1 crt2 crt3 triple mutation background had an adverse effect on pollen viability and pollen tube growth, leading to a significant reduction in fertility. The cnx1 crt1 crt2 crt3 quadruple mutation also conferred severe defects in growth and development, including a shortened primary root, increased root hair length and density, and reduced plant height. Disruption of all five members of the CNX/CRT family was revealed to be lethal. Finally, the abnormal phenotype of the cnx1 crt1 crt2 crt3 quadruple mutants was completely rescued by either the CNX1 or CNX2 cDNA under the control of the CNX1 promoter, suggesting functional redundancy between CNX1 and CNX2. Taken together, these results provide genetic evidence that CNX and CRT play essential and overlapping roles during vegetative growth and male gametophyte development in Arabidopsis.

Characterization of Brassica napus Flavonol Synthase Involved in Flavonol Biosynthesis in Brassica napus L.

Recently, Brassica napus has become a very important crop for plant oil production. Flavonols, an uncolored flavonoid subclass, have a high antioxidative effect and are known to have antiproliferative, antiangiogenic, and neuropharmacological properties. In B. napus, some flavonoid structural genes have been identified, such as, BnF3H-1, BnCHS, and BnC4H-1. However, no studies on FLS genes in B. napus have been conducted. Thus, in this study, we cloned and characterized the function of BnFLS gene B. napus. By overexpression of the BnFLS gene, flavonol (kaempferol and quercetin) levels were recovered in the Arabidopsis atfls1-ko mutant. In addition, we found that the higher endogenous flavonol levels of BnFLS-ox in vitro shoots correlated with slightly higher ROS scavenging activities. Thus, our results indicate that the BnFLS gene encodes for a BnFLS enzyme that can be manipulated to specifically increase flavonol accumulation in oilseed plants and other species such as Arabidopsis.