The Plant Journal

Del Vecchio, Hernan A.; Ying, Sheng; Park, Joonho; Knowles, Vicki L.; Kanno, Satomi; Tanoi, Keitaro; She, Yi‐Min; Plaxton, William C.

doi: 10.1111/tpj.12663pmid: 25270985

Plant purple acid phosphatases (PAPs) belong to a relatively large gene family whose individual functions are poorly understood. Three PAP isozymes that are up‐regulated in the cell walls of phosphate (Pi)‐starved (−Pi) Arabidopsis thaliana suspension cells were purified and identified by MS as AtPAP12 (At2g27190), AtPAP25 (At4g36350) and AtPAP26 (At5g34850). AtPAP12 and AtPAP26 were previously isolated from the culture medium of −Pi cell cultures, and shown to be secreted by roots of Arabidopsis seedlings to facilitate Pi scavenging from soil‐localized organophosphates. AtPAP25 exists as a 55 kDa monomer containing complex NX(S/T) glycosylation motifs at Asn172, Asn367 and Asn424. Transcript profiling and immunoblotting with anti‐AtPAP25 immune serum indicated that AtPAP25 is exclusively synthesized under −Pi conditions. Coupled with potent mixed‐type inhibition of AtPAP25 by Pi (I50 = 50 μm), this indicates a tight feedback control by Pi that prevents AtPAP25 from being synthesized or functioning as a phosphatase except when Pi levels are quite low. Promoter–GUS reporter assays revealed AtPAP25 expression in shoot vascular tissue of −Pi plants. Development of an atpap25 T‐DNA insertion mutant was arrested during cultivation on soil lacking soluble Pi, but rescued upon Pi fertilization or complementation with AtPAP25. Transcript profiling by quantitative RT‐PCR indicated that Pi starvation signaling was attenuated in the atpap25 mutant. AtPAP25 exhibited near‐optimal phosphatase activity with several phosphoproteins and phosphoamino acids as substrates. We hypothesize that AtPAP25 plays a key signaling role during Pi deprivation by functioning as a phosphoprotein phosphatase rather than as a non‐specific scavenger of Pi from extracellular P‐monoesters.

Xu, Yingxiu; Du, Jianchang

doi: 10.1111/tpj.12656pmid: 25182777

Long terminal repeat (LTR) retrotransposons are the major DNA components of flowering plants. They are generally enriched in pericentromeric heterochromatin regions of their host genomes, which could result from the preferential insertion of LTR retrotransposons and the low effectiveness of purifying selection in these regions. To estimate the relative importance of the actions of these two factors on their distribution pattern, the LTR retrotransposons in Solanum lycopersicum (tomato) plants were characterized at the genome level, and then the distribution of young elements was compared with that of relatively old elements. The current data show that old elements are mainly located in recombination‐suppressed heterochromatin regions, and that young elements are preferentially located in the gene‐rich euchromatic regions. Further analysis showed a negative correlation between the insertion time of LTR retrotransposons and the recombination rate. The data also showed there to be more solo LTRs in genic regions than in intergenic regions or in regions close to genes. These observations indicate that, unlike in many other plant genomes, the current LTR retrotransposons in tomatoes have a tendency to be preferentially located into euchromatic regions, probably caused by their severe suppression of activities in heterochromatic regions. These elements are apt to be maintained in heterochromatin regions, probably as a consequence of the pericentromeric effect in tomatoes. These results also indicate that local recombination rates and intensities of purifying selection in different genomic regions are largely responsible for structural variation and non‐random distribution of LTR retrotransposons in tomato plants.

Midorikawa, Takafumi; Endow, Joshua K.; Dufour, Jeremy; Zhu, Jieping; Inoue, Kentaro

doi: 10.1111/tpj.12655pmid: 25182596

Thylakoids are the photosynthetic membranes in chloroplasts and cyanobacteria. The aqueous phase inside the thylakoid known as the thylakoid lumen plays an essential role in the photosynthetic electron transport. The presence and significance of thiol‐disulfide exchange in this compartment have been recognized but remain poorly understood. All proteins found free in the thylakoid lumen and some proteins associated to the thylakoid membrane require an N‐terminal targeting signal, which is removed in the lumen by a membrane‐bound serine protease called thylakoidal processing peptidase (TPP). TPP is homologous to Escherichia coli type I signal peptidase (SPI) called LepB. Genetic data indicate that plastidic SPI 1 (Plsp1) is the main TPP in Arabidopsis thaliana (Arabidopsis) although biochemical evidence had been lacking. Here we demonstrate catalytic activity of bacterially produced Arabidopsis Plsp1. Recombinant Plsp1 showed processing activity against various TPP substrates at a level comparable to that of LepB. Plsp1 and LepB were also similar in the pH optima, sensitivity to arylomycin variants and a preference for the residue at −3 to the cleavage site within a substrate. Plsp1 orthologs found in angiosperms contain two unique Cys residues located in the lumen. Results of processing assays suggested that these residues were redox active and formation of a disulfide bond between them was necessary for the activity of recombinant Arabidopsis Plsp1. Furthermore, Plsp1 in Arabidopsis and pea thylakoids migrated faster under non‐reducing conditions than under reducing conditions on SDS‐PAGE. These results underpin the notion that Plsp1 is a redox‐dependent signal peptidase in the thylakoid lumen.

Pitaksaringkarn, Weerasak; Matsuoka, Keita; Asahina, Masashi; Miura, Kenji; Sage‐Ono, Kimiyo; Ono, Michiyuki; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Ishii, Tadashi; Iwai, Hiroaki; Satoh, Shinobu

doi: 10.1111/tpj.12654

Zhao, Lihua; He, Jiangman; Cai, Hanyang; Lin, Haiyan; Li, Yanqiang; Liu, Renyi; Yang, Zhenbiao; Qin, Yuan

doi: 10.1111/tpj.12657pmid: 25182975

Megasporogenesis is essential for female fertility, and requires the accomplishment of meiosis and the formation of functional megaspores. The inaccessibility and low abundance of female meiocytes make it particularly difficult to elucidate the molecular basis underlying megasporogenesis. We used high‐throughput tag‐sequencing analysis to identify genes expressed in female meiocytes (FMs) by comparing gene expression profiles from wild‐type ovules undergoing megasporogenesis with those from the spl mutant ovules, which lack megasporogenesis. A total of 862 genes were identified as FMs, with levels that are consistently reduced in spl ovules in two biological replicates. Fluorescence‐assisted cell sorting followed by RNA‐seq analysis of DMC1:GFP‐labeled female meiocytes confirmed that 90% of the FMs are indeed detected in the female meiocyte protoplast profiling. We performed reverse genetic analysis of 120 candidate genes and identified four FM genes with a function in female meiosis progression in Arabidopsis. We further revealed that KLU, a putative cytochrome P450 monooxygenase, is involved in chromosome pairing during female meiosis, most likely by affecting the normal expression pattern of DMC1 in ovules during female meiosis. Our studies provide valuable information for functional genomic analyses of plant germline development as well as insights into meiosis.

Liu, Xigang; Dinh, Thanh Theresa; Li, Dongming; Shi, Bihai; Li, Yongpeng; Cao, Xiuwei; Guo, Lin; Pan, Yanyun; Jiao, Yuling; Chen, Xuemei

doi: 10.1111/tpj.12658pmid: 25187180

In Arabidopsis, AUXIN RESPONSE FACTOR 3 (ARF3) belongs to the auxin response factor (ARF) family that regulates the expression of auxin‐responsive genes. ARF3 is known to function in leaf polarity specification and gynoecium patterning. In this study, we discovered a previously unknown role for ARF3 in floral meristem (FM) determinacy through the isolation and characterization of a mutant of ARF3 that enhanced the FM determinacy defects of agamous (ag)‐10, a weak ag allele. Central players in FM determinacy include WUSCHEL (WUS), a gene critical for FM maintenance, and AG and APETALA2 (AP2), which regulate FM determinacy by repression and promotion of WUS expression, respectively. We showed that ARF3 confers FM determinacy through repression of WUS expression, and associates with the WUS locus in part in an AG‐dependent manner. We demonstrated that ARF3 is a direct target of AP2 and partially mediates AP2's function in FM determinacy. ARF3 exhibits dynamic and complex expression patterns in floral organ primordia; altering the patterns spatially compromised FM determinacy. This study uncovered a role for ARF3 in FM determinacy and revealed relationships among genes in the genetic network governing FM determinacy.

Desai, Mintu; Rangarajan, Padma; Donahue, Janet L.; Williams, Sarah P.; Land, Eric S.; Mandal, Mihir K.; Phillippy, Brian Q.; Perera, Imara Y.; Raboy, Victor; Gillaspy, Glenda E.

doi: 10.1111/tpj.12669pmid: 25231822

Feng, Cui‐Zhu; Chen, Yun; Wang, Cun; Kong, You‐Han; Wu, Wei‐Hua; Chen, Yi‐Fang

doi: 10.1111/tpj.12670pmid: 25231920

The phytohormone abscisic acid (ABA) modulates a number of processes during plant growth and development. In this study, the molecular mechanism of Arabidopsis RAV (Related to ABI3/VP1) transcription factor RAV1 involving ABA signaling was investigated. RAV1‐underexpressing lines were more sensitive to ABA than wild‐type plants during seed germination and early seedling development, whereas RAV1‐overexpressing lines showed strong ABA‐insensitive phenotypes. Overexpression of RAV1 repressed ABI3, ABI4, and ABI5 expression, and RAV1 bound to the ABI3, ABI4, and ABI5 promoters in vitro and in vivo, indicating that RAV1 directly down‐regulates the expression of ABI3, ABI4, and ABI5. The interruption of ABI5 function in RAV1‐U abi5 plants abolished the ABA‐hypersensitive phenotype of RAV1‐U plants, demonstrating that ABI5 is epistatic to RAV1. RAV1 interacted with SNF1‐RELATED PROTEIN KINASE SnRK2.2, SnRK2.3 and SnRK2.6 in the nucleus. In vitro kinase assays showed that SnRK2.2, SnRK2.3 and SnRK2.6 phosphorylated RAV1. Transient expression assays revealed that SnRK2.2, SnRK2.3 and SnRK2.6 reduced the RAV1‐dependent repression of ABI5, and the ABA‐insensitive phenotype of the RAV1‐overexpressing line was impaired by overexpression of SnRK2.3 in the RAV1 OE3 plants. Together, these results demonstrated that the Arabidopsis RAV1 transcription factor plays an important role in ABA signaling by modulating the expression of ABI3, ABI4, and ABI5, and that its activity is negatively affected by SnRK2s.

Chen, Ke; Dorlhac de Borne, François; Szegedi, Ernö; Otten, Léon

doi: 10.1111/tpj.12661pmid: 25219519

Nicotiana species carry cellular T‐DNA sequences (cT‐DNAs), acquired by Agrobacterium‐mediated transformation. We characterized the cT‐DNA sequences of the ancestral Nicotiana tabacum species Nicotiana tomentosiformis by deep sequencing. N. tomentosiformis contains four cT‐DNA inserts derived from different Agrobacterium strains. Each has an incomplete inverted‐repeat structure. TA is similar to part of the Agrobacterium rhizogenes 1724 mikimopine‐type T‐DNA, but has unusual orf14 and mis genes. TB carries a 1724 mikimopine‐type orf14‐mis fragment and a mannopine‐agropine synthesis region (mas2‐mas1‐ags). The mas2′ gene codes for an active enzyme. TC is similar to the left part of the A. rhizogenes A4 T‐DNA, but also carries octopine synthase‐like (ocl) and c‐like genes normally found in A. tumefaciens. TD shows a complex rearrangement of T‐DNA fragments similar to the right end of the A4 TL‐DNA, and including an orf14‐like gene and a gene with unknown function, orf511. The TA, TB, TC and TD insertion sites were identified by alignment with N. tabacum and Nicotiana sylvestris sequences. The divergence values for the TA, TB, TC and TD repeats provide an estimate for their relative introduction times. A large deletion has occurred in the central part of the N. tabacum cv. Basma/Xanthi TA region, and another deletion removed the complete TC region in N. tabacum. Nicotiana otophora lacks TA, TB and TD, but contains TC and another cT‐DNA, TE. This analysis, together with that of Nicotiana glauca and other Nicotiana species, indicates multiple sequential insertions of cT‐DNAs during the evolution of the genus Nicotiana.

Ham, Byung‐Kook; Li, Gang; Jia, Weitao; Leary, Julie A.; Lucas, William J.

doi: 10.1111/tpj.12662pmid: 25227635

In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense‐related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL‐RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem‐localized protein kinase, PSRPK1. During long‐distance transport, PSRP1–sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1–sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants.