SUMMARYLEAFY COTYLEDON 1 (LEC1) plays vital roles in the regulation of seed maturation in Arabidopsis. LEC1 encodes a homolog of yeast HAP3 or mammalian NF-YB/CBF-A subunit of trimeric CCAAT binding factor (CBF). Among the nine paralogs of NF-YB in Arabidopsis, LEC1-LIKE (L1L) is most closely related to LEC1, and can complement the lec1 mutation when expressed under the control of the LEC1 promoter. Although the nature of the B3-type seed maturation regulators as transcription factors have been investigated, knowledge of the molecular action of LEC1 is limited. When co-expressed with NF-YC2 in the presence of ABA, we found that LEC1 or L1L, but not other NF-YBs, activated the promoter of CRUCIFERIN C (CRC), which encodes a seed storage protein. However, additional expression of an NF-YA subunit interfered with the activation. The LEC1/ L1L-[NF-YC2] activation depended on ABA-response elements present in the promoter, which led to the finding that LEC1/L1L-[NF-YC2] can strongly activate the CRC promoter in the absence of ABA when co-expressed with a seed-specific ABA-response element (ABRE)-binding factor, bZIP67. Functional coupling of LEC1/L1L-[AtNF-YC2] and bZIP67 was also observed in the regulation of sucrose synthase 2 (SUS2). Immunoprecipitation experiments revealed that L1L and bZIP67 formed a protein complex in vivo. These results demonstrate a novel plant-specific mechanism for NF-Y subunit function that enables LEC1 and L1L to regulate a defined developmental network.

Arabidopsis ABSCISIC ACID INSENSEITIVE3 (ABI3), FUSCA3 (FUS3) and LEAFY COTYLEDON1 (LEC1) encode key transcription factors that control seed maturation events, including seed storage protein (SSP) accumulation. Although lec1 mutations are known to down-regulate SSP gene expression as the fus3 or abi3 mutation does, the mechanisms by which LEC1 regulates SSP gene expression are largely unknown compared with the mechanisms utilized by FUS3 or ABI3. We expressed LEC1 ectopically in transgenic plants using an artificial dexamethasone (Dex) induction system. The ectopic expression of LEC1 also resulted in the induction of FUS3 and ABI3 expression, which preceded the induction of SSP expression. The expression of FUS3 and ABI3 was found to be down-regulated in developing siliques of the lec1 mutant. Furthermore, the levels of ectopic SSP induction by LEC1 were greatly or moderately reduced in transgenic plants with an abi3 or fus3 mutant background, respectively. LEC1-induced ectopic expression of the At1g62290 aspartic protease gene, which was identified to be regulated preferentially by FUS3, was more severely affected in the fus3 mutant than in the abi3 mutant. From these data, we suggest that LEC1 controls the expression of the SSP genes in a hierarchical manner, which involves ABI3 and FUS3.

The key transcription factors that control seed maturation, ABSCISIC ACID INSENSITIVE3 (ABI3) and FUSCA3 (FUS3), share homologous DNA-binding domains. Regulation of seed storage protein genes At2S3 and CRC by ABI3 and FUS3 was investigated using transgenic plants in which ABI3 and FUS3 could be ectopically induced by steroid hormones. Like ABI3, the presence of FUS3 led to expression of At2S3 and CRC in vegetative tissues. FUS3-mediated induction of CRC was completely dependent on exogenous abscisic acid (ABA), while At2S3 was weakly induced without ABA but strongly enhanced with ABA. This ABA dependency of FUS3-induced CRC and At2S3 expression was similar to that observed for ABI3. However, kinetic analysis revealed distinctions between the mechanisms of ABA-dependent CRC regulation by FUS3 or ABI3, and between target genes. While At2S3 activation by FUS3 was rapid, CRC induction by FUS3 in the presence of ABA, and by ABA followed by the presence of FUS3, took a significantly longer time (24-36 h). This suggested the involvement of an indirect mechanism requiring the ABA- and FUS3-dependent synthesis of intermediate regulatory factor(s). A chimeric protein composed of the FUS3 B3 domain, and a heterologous activation domain and nuclear localization signal exhibited a tight coupling with ABA regulation as observed for wild-type FUS3. Simultaneous induction of FUS3 and ABI3 did not result in the synergistic activation of CRC and At2S3. Based on these results, similarities and differences in the mechanisms of seed storage protein gene regulation by FUS3 and ABI3 are discussed.