In Vitro Enzyme Activity of Recombinant Chlase Versions.
Citrus Chlase versions were expressed as recombinant proteins in E. coli. Total soluble proteins were extracted from E. coli harboring pTrxFus plasmid expressing thioredoxin (pTrx; 12 kD), pTrxFus-Chlase plasmid containing the mature form of Chlase (lacking the N-terminal 21 amino acids) fused to thioredoxin (pTrx-ChlaseΔN; 45 kD), or pTrxFus-Chlase-S147T plasmid containing a version of ChlaseΔN bearing a Ser-to-Thr point mutation at position 147 (pTrx-ChlaseΔN-S147T; 45 kD). Protein extracts, and a protein-free reaction control, were examined for Chlase in vitro activity (A) as described in Methods and for Chlase-thioredoxin recombinant fusion protein level by protein gel blot analysis (B) using thioredoxin-specific antibodies.

Phenotypes of Plants Overexpressing Chlase Versions under Low-Intensity Light.
(A) Squash plants were infected with empty viral vector (AGII), viral vector harboring the full-length Chlase gene (AGII-Chlase), or viral vector harboring the N-terminally deleted form of the Chlase gene (AGII-ChlaseΔN). Plants were grown for 10 d after inoculation under low-intensity fluorescent light (15 μE), and leaf 3 of each plant was documented.
(B) Kinetics of in vitro Chlase activity was measured from crude extracts of leaf 4 of the plants used for (A), as described in Methods. Data points represent AGII-Chlase (squares), AGII-ChlaseΔN (triangles), and AGII (circles).
(C) Squash plants were infected with viral vector harboring the N-terminally deleted form of the Chlase gene in the wild-type form (AGII-ChlaseΔN) or mutated at Ser-147 to encode Thr (AGII-ChlaseΔN S147T). Plants were grown for 10 d after inoculation under low-intensity fluorescent light (15 μE), and leaf 3 of each plant was documented.

Phenotypes of Plants Overexpressing Chlase Versions under Natural Light.
(A) Squash plants were infected with empty viral vector (AGII), viral vector harboring the full-length Chlase gene (AGII-Chlase), or viral vector harboring the N-terminally deleted form of the Chlase gene (AGII-ChlaseΔN). Plants were grown for 10 d after inoculation under natural light in a greenhouse (500 μE), and leaf 3 of each plant was documented.
(B) Kinetics of in vitro Chlase activity was measured in crude extracts of leaf 4 of the plants used for (A), as described in Methods. Data points represent full-length AGII-Chlase (squares), N-terminally deleted AGII-ChlaseΔN (triangles), and vector control AGII (circles).
(C) Squash plants were infected with viral vector harboring the N-terminally deleted form of the Chlase gene in the wild-type form (AGII-ChlaseΔN) or mutated at Ser-147 to encode Thr (AGII-ChlaseΔN S147T). Plants were grown for 10 d after inoculation under natural light in a greenhouse (500 μE), and leaf 3 of each plant was documented. Blow-ups of the leaves (insets) enable a more detailed perspective of the leaf surface and phenotype.

Relative Chlorophyll Levels, Chlase Processing, and Localization in Tobacco Protoplasts Transiently Expressing Citrus Chlase Versions.
(A) Tobacco protoplasts were transiently transformed with plasmids harboring the gene encoding GFP alone (p35S-GFP) or in combination with either full-length Chlase (p35S-Chlase+35S-GFP) or N-terminally deleted Chlase (p35S-ChlaseΔN+35S-GFP).
(B) After 72 h of transient expression, fluorescence was visualized using a laser scanning confocal microscope as described in Methods. One representative protoplast for each construct is visually presented as follows: panel 1, green fluorescence corresponds to GFP; panel 2, red fluorescence corresponds to chlorophyll; panel 3, confocal image recorded simultaneously in transmitted and red fluorescence mode (i.e., chlorophyll fluorescence overlaid on the bright-field image); and panel 4, confocal image recorded simultaneously for red and green fluorescence (i.e., GFP and chlorophyll fluorescence overlaid). Protoplasts displaying similar levels of GFP fluorescence were selected for semiquantitative analysis of chlorophyll relative levels by fluorescence. Average fluorescence measured for protoplasts expressing GFP alone (CAI = 15.55 ± 1.8; n = 3) was designated as 100%. Average fluorescence for protoplasts expressing Chlase with GFP (CAI = 3.6 ± 0.5; n = 3) and protoplasts expressing ChlaseΔN with GFP (CAI = 0.71 ± 0.64; n = 5) are depicted as percentages relative to protoplasts expressing GFP alone.
(C) Analysis of expression and processing kinetics of citrus Chlase versions in protoplasts. Tobacco protoplasts were transiently transformed with plasmids directing coexpression of Chlase and GFP (p35S-Chlase+35S-GFP), coexpression of ChlaseΔN and GFP (p35S-ChlaseΔN+35S-GFP), or GFP alone as a control. Protoplasts were harvested at 24, 48, and 72 h after transformation, and total proteins extracted with USB protein extraction buffer were separated by SDS-PAGE (30 μg per sample), blotted, and dressed with anti-citrus Chlase antibodies for the detection of citrus Chlase versions. Protoplasts expressing GFP alone served as a negative control for citrus Chlase–specific detection.
(D) Intracellular localization of Chlase versions in protoplasts. Tobacco protoplasts transiently transformed with the constructs mentioned above were harvested at 72 h after transformation. Intact chloroplasts were isolated from lysed protoplasts by Percoll gradient centrifugation and were in turn lysed and fractionated to membrane and soluble fractions. Membrane-associated proteins were washed with buffer containing 200 mM NaCl (data not shown) followed by extraction with buffer containing 0.5% Triton X-100. Proteins were precipitated with acetone and resuspended in USB protein extraction buffer from intact protoplasts, intact chloroplasts, chloroplast soluble and membrane fractions, 0.5% Triton X-100 wash of membrane fractions, and membrane pellets after Triton wash. Proteins were separated by SDS-PAGE (loading based on equal chlorophyll: 6 μg per lane), blotted, and dressed with anti-citrus Chlase antibodies for the detection of expressed Chlase versions, anti-LHCII (thylakoid integral membrane protein) antibodies, and anti-OE33 (lumen protein) antibodies for the detection of the endogenous proteins that serve as controls. The GFP control lane contains total protein extract from transformed protoplasts expressing GFP but not expressing any of the citrus Chlase versions.

Chlorophyll Catabolic Pathway.
The first four enzymatic steps of the chlorophyll catabolic pathway leading to loss of the typical green color are outlined. Structures of chlorophyll and the intermediate breakdown products are shown. Enzyme-catalyzed chlorophyll breakdown steps are as follows. (1) Chlase, an esterase, catalyzes the cleavage of the hydrophobic thylakoid-anchoring phytol chain of chlorophyll from the porphyrin ring, resulting in the product chlorophyllide, which retains the typical green color. (2) Mg-dechelatase is responsible for removing the Mg ion from chlorophyllide to yield pheophorbide a, which retains the green color. (3) Pheophorbide a oxygenase (PaO) catalyzes the cleavage of the porphyrin ring, resulting in the red chlorophyll catabolite (RCC), which is further reduced by the enzyme red chlorophyll catabolite reductase (RCCR) to a primary fluorescent chlorophyll catabolite (pFCC). pFCCs are further metabolized by additional enzymes downstream and exported to the vacuole, where they are nonenzymatically converted to nonfluorescent chlorophyll catabolites (NCCs) and stored indefinitely.

Construction and Expression of a Viral Vector Containing the Citrus Chlase1 Gene.
(A) Scheme of the ZYMV-based AGII viral vector. AGII noncoding (hatched boxes) and coding (open boxes) regions, including the foreign gene (FG) cloning site, are shown. The sequence blow-up indicates the NIa protease cleavage sites involved in proteolysis of the foreign gene product from the viral polyprotein. Amino acid sequences corresponding to the NIa protease recognition motif are indicated in boldface, and cleavage sites are indicated by slashes interrupting the sequence and by the arrows. An extension of six amino acids added to the C terminus of Chlase versions (after processing) is underlined. P1, first protein; HC-Pro, helper component-proteinase; P3, third protein; CI, cytoplasmic inclusion; NIa, nuclear inclusion a; NIb, nuclear inclusion b; CP, coat protein; FG, foreign gene.
(B) RT-PCR analysis of viral RNA from infected plants. Total RNA was extracted from noninfected squash leaves (virus free) or from squash leaves systemically infected with empty viral vector (AGII), viral vector harboring full-length Chlase (AGII-Chlase), or N-terminally deleted Chlase (AGII-ChlaseΔN) at 8 and 21 d after inoculation (dpi). RNA was subjected to RT-PCR with primers flanking the foreign gene (FG) insertion site (see arrows in [A]). Amplified products were separated by gel electrophoresis, together with molecular weight markers (M), and stained with ethidium bromide.

Dynamics of the Chlorotic Phenotype in ChlaseΔN-Expressing Plants.
Phenotype was monitored and documented once every 2 d for leaf 4 of squash plants infected with the viral vector harboring the N-terminally deleted Chlase gene (AGII-ChlaseΔN). Phenotypes shown were documented for the same leaf on days 8 to 16 after viral vector inoculation (dpi).

The Lesion-Mimic Phenotype Caused by ChlaseΔN Overexpression Is Light-Dependent and Results from the Accumulation of Chlorophyll Breakdown Product.
(A) Plants infected with viral vector harboring the N-terminally deleted Chlase gene (AGII-ChlaseΔN) were grown under natural light conditions so that half of leaf 3 was exposed and half was shaded by black netting, blocking ∼90% of the light. The displayed leaf was documented at 10 d after inoculation.
(B) Leaves (leaf 3) weighing ∼100 mg were collected from noninfected plants and from plants infected with empty viral vector (AGII), viral vector harboring the full-length Chlase gene (AGII-Chlase), or viral vector harboring the N-terminally deleted form of the Chlase gene (AGII-ChlaseΔN). Chlorophylls were extracted from pools of five leaves each, and chlorophyll and chlorophyllide were separated by an acetone/hexane phase separation assay as described in Methods. The ratio of chlorophyllide to chlorophyll is displayed visually (phase separations at bottom) and as a histogram (top) showing the spectral quantitation results separately for chlorophyllide a/chlorophyll a (black columns) and chlorophyllide b/chlorophyll b (white columns).